Optical system and assay chip for probing, detecting and analyzing molecules

ABSTRACT

Apparatus and methods for analyzing single molecules and performing nucleic acid sequencing. An apparatus can include an assay chip that includes multiple pixels with sample wells configured to receive a sample, which, when excited, emits emission energy; at least one element for directing the emission energy in a particular direction; and a light path along which the emission energy travels from the sample well toward a sensor. The apparatus also includes an instrument that interfaces with the assay chip. The instrument includes an excitation light source for exciting the sample in each sample well; a plurality of sensors corresponding the sample wells. Each sensor may detect emission energy from a sample in a respective sample well. The instrument includes at least one optical element that directs the emission energy from each sample well towards a respective sensor of the plurality of sensors.

RELATED APPLICATIONS

This application is a continuation and claims the benefit under 35U.S.C. § 120 of U.S. application Ser. No. 15/882,776, filed on Jan. 29,2018, which is hereby incorporated by reference in its entirety.Application Ser. No. 15/882,776 is a continuation and claims the benefitunder 35 U.S.C. § 120 of U.S. application Ser. No. 14/821,686 filed onAug. 7, 2015, which is hereby incorporated by reference in its entirety.Application Ser. No. 14/821,686 claims priority to U.S. ProvisionalPatent Application No. 62/035,242 filed Aug. 8, 2014, which is herebyincorporated by reference in its entirety.

This application is related to the following U.S. applications:

-   -   U.S. Provisional Patent Application 62/164,506, entitled        “INTEGRATED DEVICE FOR TEMPORAL BINNING OF RECEIVED PHOTONS”,        filed on May 20, 2015;    -   U.S. Provisional Patent Application 62/164,485, entitled “PULSED        LASER”, filed on May 20, 2015;    -   U.S. Provisional Patent Application 62/164,482, entitled        “METHODS FOR NUCLEIC ACID SEQUENCING”, filed on May 20, 2015;    -   U.S. Provisional Patent Application 62/164,464, entitled        “INTEGRATED DEVICE WITH EXTERNAL LIGHT SOURCE FOR PROBING        DETECTING AND ANALYZING MOLECULES”, filed on May 20, 2015;    -   A U.S. non-provisional patent application Ser. No. 14/821,656,        filed on Aug. 7, 2015, bearing docket no. R0708.70002US02,        titled “INTEGRATED DEVICE FOR TEMPORAL BINNING OF RECEIVED        PHOTONS;” and    -   A U.S. non-provisional patent application Ser. No. 14/821,688        filed on Aug. 7, 2015, bearing docket no. R0708.70004US02,        titled “INTEGRATED DEVICE WITH EXTERNAL LIGHT SOURCE FOR        PROBING, DETECTING, AND ANALYZING MOLECULES.”

Each of the above-listed related applications is hereby incorporated byreference in its entirety.

BACKGROUND Field

The present application is directed generally to devices, methods andtechniques for performing rapid, massively parallel, quantitativeanalysis of biological and/or chemical samples, and methods offabricating said devices.

Related Art

Detection and analysis of biological samples may be performed usingbiological assays (“bioassays”). Bioassays conventionally involve large,expensive laboratory equipment requiring research scientists trained tooperate the equipment and perform the bioassays. Moreover, bioassays areconventionally performed in bulk such that a large amount of aparticular type of sample is necessary for detection and quantitation.

Some bioassays are performed by tagging samples with luminescent tagsthat emit light of a particular wavelength. The tags are illuminatedwith an excitation light source to cause luminescence, and theluminescent light is detected with a photodetector to quantify theamount of luminescent light emitted by the tags. Bioassays usingluminescent tags conventionally involve expensive laser light sources toilluminate samples and complicated, bulky detection optics andelectronics to collect the luminescence from the illuminated samples.

SUMMARY

Some embodiments relate to an instrument configured to interface with anassay chip that includes a plurality of sample wells. Each sample wellof the plurality of sample wells may receive a sample. The instrumentincludes at least one pulsed excitation light source for exciting thesample of at least a portion of the plurality of sample wells. Theinstrument also includes a plurality of sensors, each sensor of theplurality of sensors corresponding to a sample well of the plurality ofsample wells. Each sensor of the plurality of sensors is detectsemission energy from the sample in a respective sample well. Also, eachsensor of the plurality of sensors is capable of detecting the detectiontime of the emission energy. The instrument also includes at least oneoptical element configured to direct the emission energy from eachsample well of the plurality of sample wells towards a respective sensorof the plurality of sensors.

Some embodiments relate to an apparatus that includes an assay chip andan instrument. The assay chip includes a plurality of pixels. Each pixelof the assay chip includes a sample well that receives a sample, which,when excited, emits emission energy. Each pixel of the assay chip alsoincludes at least one element for directing the emission energy in aparticular direction. The at least one element may be a refractiveelement, a diffractive element, a plasmonic element or a resonator. Eachpixel of the assay chip also includes a light path along which theemission energy travels from the sample well toward a sensor of theinstrument. The instrument interfaces with the assay chip and includesat least one pulsed excitation light source that excites the sample ineach sample well. The instrument also includes a plurality of sensors,each sensor of the plurality of sensors corresponding to a respectivesample well. Each sensor of the plurality of sensors is detects emissionenergy from the sample in the respective sample well and detects thedetection time of the emission energy. The instrument also includes atleast one optical element that directs the emission energy from eachsample well towards a respective sensor of the plurality of sensors.

Some embodiments relate to a method of analyzing a specimen. The methodincludes providing the specimen on the top surface of an assay chip thatincludes a plurality of sample wells. The method also includes aligningthe chip with an instrument that includes at least one excitation lightsource and at least one sensor. The method also includes exciting asample from the specimen in at least one of the plurality of samplewells with pulsed excitation light from the at least one pulsedexcitation light source. The method also includes detecting, with the atleast one sensor, emission energy generated by the sample in the atleast one sample well in response to excitation by the excitation light.The at least one sensor is capable of determining the lifetime of theemission energy generated by the sample.

Some embodiments relate to a method for sequencing a target nucleic acidmolecule. The method includes providing a chip adjacent to an instrumentthat includes a pulsed excitation source and a sensor capable ofdetecting at least one temporal property of light. The chip includes atleast one well that is operatively coupled to said excitation source andsaid sensor when said chip is at a sensing position of said instrument.The well contains said target nucleic acid molecule, a polymerizingenzyme and a plurality of types of nucleotides or nucleotide analogs.The method also includes, with said chip at said sensing position,performing an extension reaction at a priming location of said targetnucleic acid molecule in the presence of said polymerizing enzyme tosequentially incorporate said nucleotides or nucleotide analogs into agrowing strand that is complementary to said target nucleic acidmolecule. Upon incorporation and excitation by excitation energy fromsaid excitation source, said nucleotides or nucleotides analogs emitsignals in said well. The method also includes using said sensor todetect spatial and/or temporal distribution patterns of said signalsthat are distinguishable for said plurality of types of nucleotides ornucleotide analogs. The method also includes identifying saidnucleotides or nucleotide analogs based on said spatial and/or temporaldistribution patterns of said signals, thereby sequencing said targetnucleic acid molecule.

Some embodiments relate to a method for nucleic acid sequencing. Themethod includes providing a chip adjacent to an instrument. The chipincludes a plurality of wells that are each operatively coupled to apulsed excitation source and a sensor of said instrument when said chipis at a sensing position of said instrument. An individual well of saidplurality of wells contains said target nucleic acid molecule, apolymerizing enzyme and a plurality of types of nucleotides ornucleotide analogs. The method also includes, with the chip at saidsensing position, subjecting said target nucleic acid molecule to apolymerization reaction to yield a growing strand that is complementaryto said target nucleic acid molecule in the presence of said nucleotidesor nucleotide analogs and said polymerizing enzyme. The nucleotides ornucleotides analogs emit signals in said individual well upon excitationby excitation energy from said excitation source during incorporation.The method also includes using said sensor to detect temporaldistribution patterns of said signals that are distinguishable for saidplurality of types of nucleotides or nucleotide analogs. The method alsoincludes identifying a sequence of said target nucleic acid moleculebased on said spatial and/or temporal distribution patterns of saidsignals.

The foregoing and other aspects, embodiments, and features of thepresent teachings can be more fully understood from the followingdescription in conjunction with the accompanying drawings.

The term “pixel” may be used in the present disclosure to refer to aunit cell of an integrated device. The unit cell may include a samplewell and a sensor. The unit cell may further include an excitationsource. The unit cell may further include at least oneexcitation-coupling optical structure (which may be referred to as a“first structure”) that is configured to enhance coupling of excitationenergy from the excitation source to the sample well. The unit cell mayfurther include at least one emission-coupling structure that isconfigured to enhance coupling of emission from the sample well to thesensor. The unit cell may further include integrated electronic devices(e.g., CMOS devices). There may be a plurality of pixels arranged in anarray on an integrated device.

The term “optical” may be used in the present disclosure to refer tovisible, near infrared, and short-wavelength infrared spectral bands.

The term “tag” may be used in the present disclosure to refer to a tag,probe, marker, or reporter attached to a sample to be analyzed orattached to a reactant that may be reacted with a sample.

The phrase “excitation energy” may be used in the present disclosure torefer to any form of energy (e.g., radiative or non-radiative) deliveredto a sample and/or tag within the sample well. Radiative excitationenergy may comprise optical radiation at one or more characteristicwavelengths.

The phrase “characteristic wavelength” may be used in the presentdisclosure to refer to a central or predominant wavelength within alimited bandwidth of radiation. In some cases, it may refer to a peakwavelength of a bandwidth of radiation. Examples of characteristicwavelengths of fluorophores are 563 nm, 595 nm, 662 nm, and 687 nm.

The phrase “characteristic energy” may be used in the present disclosureto refer to an energy associated with a characteristic wavelength.

The term “emission” may be used in the present disclosure to refer toemission from a tag and/or sample. This may include radiative emission(e.g., optical emission) or non-radiative energy transfer (e.g., Dexterenergy transfer or Firster resonant energy transfer). Emission resultsfrom excitation of a sample and/or tag within the sample well.

The phrase “emission from a sample well” or “emission from a sample” maybe used in the present disclosure to refer to emission from a tag and/orsample within a sample well.

The term “self-aligned” may be used in the present disclosure to referto a microfabrication process in which at least two distinct elements(e.g., a sample well and an emission-coupling structure, a sample welland an excitation-source) may be fabricated and aligned to each otherwithout using two separate lithographic patterning steps in which afirst lithographic patterning step (e.g., photolithography, ion-beamlithography, EUV lithography) prints a pattern of a first element and asecond lithographic patterning step is aligned to the first lithographicpatterning step and prints a pattern of the second element. Aself-aligned process may comprise including the pattern of both thefirst and second element in a single lithographic patterning step, ormay comprise forming the second element using features of a fabricatedstructure of the first element.

The term “sensor” may be used in the present disclosure to refer to oneor more integrated circuit devices configured to sense emission from thesample well and produce at least one electrical signal representative ofthe sensed emission.

The term “nano-scale” may be used in the present disclosure to refer toa structure having at least one dimension or minimum feature size on theorder of 150 nanometers (nm) or less, but not greater than approximately500 nm.

The term “micro-scale” may be used in the present disclosure to refer toa structure having at least one dimension or minimum feature sizebetween approximately 500 nm and approximately 100 microns.

The phrase “enhance excitation energy” may be used in the presentdisclosure to refer to increasing an intensity of excitation energy atan excitation region of a sample well. The intensity may be increased byconcentrating and/or resonating excitation energy incident on the samplewell, for example. In some cases, the intensity may be increased byanti-reflective coatings or lossy layers that allow the excitationenergy to penetrate further into the excitation region of a sample well.An enhancement of excitation energy may be a comparative reference to anembodiment that does not include structures to enhance the excitationenergy at an excitation region of a sample well.

The terms “about,” “approximately,” and “substantially” may be used inthe present disclosure to refer to a value, and are intended toencompass the referenced value plus and minus acceptable variations. Theamount of variation could be less than 5% in some embodiments, less than10% in some embodiments, and yet less than 20% in some embodiments. Inembodiments where an apparatus may function properly over a large rangeof values, e.g., a range including one or more orders of magnitude, theamount of variation could be a factor of two. For example, if anapparatus functions properly for a value ranging from 20 to 350,“approximately 80” may encompass values between 40 and 160.

The term “adjacent” may be used in the present disclosure to refer totwo elements arranged within close proximity to one another (e.g.,within a distance that is less than about one-fifth of a transverse orvertical dimension of a pixel). In some cases there may be interveningstructures or layers between adjacent elements. In some cases adjacentelements may be immediately adjacent to one another with no interveningstructures or elements.

The term “detect” may be used in the present disclosure to refer toreceiving an emission at a sensor from a sample well and producing atleast one electrical signal representative of or associated with theemission. The term “detect” may also be used in the present disclosureto refer to determining the presence of, or identifying a property of, aparticular sample or tag in the sample well based upon emission from thesample well.

BRIEF DESCRIPTION

The skilled artisan will understand that the figures, described herein,are for illustration purposes only. It is to be understood that in someinstances various aspects of the invention may be shown exaggerated orenlarged to facilitate an understanding of the invention. In thedrawings, like reference characters generally refer to like features,functionally similar and/or structurally similar elements throughout thevarious figures. The drawings are not necessarily to scale, emphasisinstead being placed upon illustrating the principles of the teachings.The drawings are not intended to limit the scope of the presentteachings in any way.

FIG. 1-1 depicts fluorescent lifetime curves for two different markers,according to some embodiments.

FIG. 1-2A depicts emission wavelength spectra, according to someembodiments.

FIG. 1-2B depicts absorption wavelength spectra, according to someembodiments.

FIG. 1-2C depicts emission wavelength spectra, according to someembodiments.

FIG. 1-3A depicts a phase space for emission wavelength and emissionlifetime.

FIG. 1-3B depicts a phase space for absorption wavelength and emissionlifetime.

FIG. 1-4 depicts a phase space for emission wavelength, absorptionwavelength, and emission lifetime.

FIG. 2-1 is a block diagram representation of an apparatus that may beused for rapid, mobile analysis of biological and chemical specimens,according to some embodiments.

FIG. 2-2 a schematic diagram of the relationship between pixels of thesensor chip and pixels of the assay chip, according to some embodiments.

FIG. 2-3 depicts components associated with a single pixel of the assaychip and a single pixel of the sensor chip, according to someembodiments.

FIG. 2-4 depicts a portion of the components of the instrument,according to some embodiments.

FIG. 3-1A is a top perspective view of the assay chip and a chip holderframe, according to some embodiments.

FIG. 3-1B is a bottom perspective view of the assay chip and the chipholder frame, according to some embodiments.

FIG. 3-1C is a cross-section view of the assay chip and the chip holderframe, according to some embodiments.

FIG. 3-2 depicts excitation energy incident on a sample well, accordingto some embodiments.

FIG. 3-3 illustrates attenuation of excitation energy along a samplewell that is formed as a zero-mode waveguide, according to someembodiments.

FIG. 3-4 depicts a sample well that includes a divot, which increasesexcitation energy at an excitation region associated with the samplewell in some embodiments.

FIG. 3-5 compares excitation intensities for sample wells with andwithout a divot, according to one embodiment.

FIG. 3-6 depicts a sample well and divot formed at a protrusion,according to some embodiments.

FIG. 3-7A depicts a sample well having tapered sidewalls, according tosome embodiments.

FIG. 3-7B depicts a sample well having curved sidewalls and a divot witha smaller transverse dimension, according to some embodiments.

FIG. 3-7C illustrates a side elevation view of a sample well formed fromsurface plasmonic structures.

FIG. 3-7D illustrates a plan view of a sample well formed from surfaceplasmonic structures.

FIG. 3-7E depicts a sample well that includes anexcitation-energy-enhancing structure formed along sidewalls of thesample well, according to some embodiments.

FIG. 3-7F depicts a sample well formed in a multi-layer stack, accordingto some embodiments.

FIG. 3-8 illustrates surface coating formed on surfaces of a samplewell, according to some embodiments.

FIG. 3-9A through FIG. 3-9E depict structures associated with a lift-offprocess of forming a sample well, according to some embodiments.

FIG. 3-9F depicts a structure associated with an alternative lift-offprocess of forming a sample well, according to some embodiments.

FIG. 3-10A through FIG. 3-10D depict structures associated with a directetching process of forming a sample well, according to some embodiments.

FIG. 3-11 depicts a sample well that may be formed in multiple layersusing a lift-off process or a direct etching process, according to someembodiments.

FIG. 3-12 depicts a structure associated with an etching process thatmay be used to form a divot, according to some embodiments.

FIG. 3-13A through FIG. 3-13C depict structures associated with analternative process of forming a divot, according to some embodiments.

FIG. 3-14A through FIG. 3-14D depict structures associated with aprocess for depositing an adherent and passivating layers, according tosome embodiments.

FIG. 3-15 depicts a structure associated with a process for depositingan adherent centrally within a sample well, according to someembodiments.

FIG. 4-1A and FIG. 4-1B depict a surface-plasmon structure, according tojust one embodiment.

FIG. 4-1C depicts a surface-plasmon structure formed adjacent a samplewell, according to some embodiments.

FIG. 4-1D and FIG. 4-1E depict surface-plasmon structures formed in asample well, according to some embodiments.

FIG. 4-2A through FIG. 4-2C depict examples of periodic surface-plasmonstructures, according to some embodiments.

FIG. 4-2D depicts a numerical simulation of excitation energy at asample well-formed adjacent a periodic surface-plasmon structure,according to some embodiments.

FIG. 4-2E through FIG. 4-2G depict periodic surface-plasmon structures,according to some embodiments.

FIG. 4-2H and FIG. 4-2I depict a nano-antenna comprising surface-plasmonstructures, according to some embodiments.

FIG. 4-3A through FIG. 4-3E depict structures associated with processsteps for forming a surface-plasmon structure, according to someembodiments.

FIG. 4-4A through FIG. 4-4G depict structures associated with processsteps for forming a surface-plasmon structure and self-aligned samplewell, according to some embodiments.

FIG. 4-5A through FIG. 4-5E depict structures associated with processsteps for forming a surface-plasmon structure and self-aligned samplewell, according to some embodiments.

FIG. 4-6A depicts a thin lossy film formed adjacent a sample well,according to some embodiments.

FIG. 4-6B and FIG. 4-6C depict results from numerical simulations ofexcitation energy in the vicinity of a sample well and thin lossy film,according to some embodiments.

FIG. 4-6D depicts a thin lossy film spaced from a sample well, accordingto some embodiments.

FIG. 4-6E depicts a thin lossy film stack formed adjacent a sample well,according to some embodiments.

FIG. 4-7A illustrates a reflective stack that may be used to form aresonant cavity adjacent a sample well, according to some embodiments.

FIG. 4-7B depicts a dielectric structure that may be used to concentrateexcitation energy at a sample well, according to some embodiments.

FIG. 4-7C and FIG. 4-7D depict a photonic bandgap structure that may bepatterned adjacent a sample well, according to some embodiments.

FIG. 4-8A through FIG. 4-8G depict structures associated with processsteps for forming dielectric structures and a self-aligned sample well,according to some embodiments.

FIG. 4-9A and FIG. 4-9B depict structures for coupling excitation energyto a sample via a non-radiative process, according to some embodiments.

FIG. 4-9C depicts a structure for coupling excitation energy to a sampleby multiple non-radiative processes, according to some embodiments.

FIG. 4-9D depicts a structure that incorporates one or moreenergy-converting particles to couple excitation energy to a sample viaa radiative or non-radiative process, according to some embodiments.

FIG. 4-9E depicts spectra associated with down conversion of excitationenergy to a sample, according to some embodiments.

FIG. 4-9F depicts spectra associated with up conversion of excitationenergy to a sample, according to some embodiments.

FIG. 5-1 depicts a concentric, plasmonic circular grating, according tosome embodiments.

FIG. 5-2 depicts a spiral plasmonic grating, according to someembodiments.

FIG. 5-3A depicts emission spatial distribution patterns from aconcentric, plasmonic circular grating, according to some embodiments.

FIG. 5-3B illustrates the directivity of aperiodic and periodicconcentric, plasmonic circular gratings, according to some embodiments.

FIG. 5-4A through FIG. 5-4B depict plasmonic nano-antennas, according tosome embodiments.

FIG. 5-5A through FIG. 5-5B depict plasmonic nano-antennas, according tosome embodiments.

FIG. 5-5C depicts the radiation pattern of emission energy from anano-antenna array, according to come embodiments.

FIG. 5-6A depicts refractive optics of the assay chip, according to someembodiments.

FIG. 5-6B depicts Fresnel lenses of the assay chip, according to someembodiments.

FIG. 6-1 depicts microscopy components of the instrument, according tosome embodiments.

FIG. 6-2A depicts far-field spectral sorting elements of the sensorchip, according to some embodiments.

FIG. 6-2B depicts far-field filtering elements of the sensor chip,according to some embodiments.

FIG. 6-3A and FIG. 6-3B depict thin lossy films of the sensor chip,according to some embodiments.

FIG. 6-4A and FIG. 6-4B depict the optical block of the instrument,according to some embodiments.

FIG. 6-5 illustrates optical paths through the optical system, accordingto some embodiments.

FIG. 7-1A depicts, in elevation view, a sensor within a pixel of asensor chip, according to some embodiments.

FIG. 7-1B depicts a bulls-eye sensor having two separate and concentricactive areas, according to some embodiments.

FIG. 7-1C depicts a stripe sensor having four separate active areas,according to some embodiments.

FIG. 7-1D depicts a quad sensor having four separate active areas,according to some embodiments.

FIG. 7-1E depicts an arc-segment sensor having four separate activeareas, according to some embodiments.

FIG. 7-1F depicts a stacked-segment sensor, according to someembodiments.

FIG. 7-2A depicts an emission distribution from the sorting elements forenergy emitted at a first wavelength, according to some embodiments.

FIG. 7-2B depicts a radiation pattern received by a bulls-eye sensorcorresponding to the emission distribution depicted in FIG. 7-2A,according to some embodiments.

FIG. 7-2C depicts an emission distribution from the sorting elements forenergy emitted at a second wavelength, according to some embodiments.

FIG. 7-2D depicts a radiation pattern received by a bulls-eye sensorcorresponding to the emission distribution depicted in FIG. 7-2C,according to some embodiments.

FIG. 7-2E represents results from a numerical simulation of signaldetection for a bulls-eye sensor having two active areas for a firstemission wavelength from a sample, according to some embodiments.

FIG. 7-2F represents results from a numerical simulation of signaldetection for the bulls-eye sensor associated with FIG. 7-2E for asecond emission wavelength from a sample, according to some embodiments.

FIG. 7-2G represents results from a numerical simulation of signaldetection for the bulls-eye sensor associated with FIG. 7-2E for a thirdemission wavelength from a sample, according to some embodiments.

FIG. 7-2H represents results from a numerical simulation of signaldetection for the bulls-eye sensor associated with FIG. 7-2E for afourth emission wavelength from a sample, according to some embodiments.

FIG. 7-2I represents results from a numerical simulation of signaldetection for a bulls-eye sensor having four active areas for a firstemission wavelength from a sample, according to some embodiments.

FIG. 7-2J represents results from a numerical simulation of signaldetection for the bulls-eye sensor associated with FIG. 7-2I for asecond emission wavelength from a sample, according to some embodiments.

FIG. 7-3A depicts circuitry on an instrument that may be used to readsignals from a sensor comprising two active areas, according to someembodiments.

FIG. 7-3B depicts a three-transistor circuit that may be included at asensor chip for signal accumulation and read-out, according to someembodiments.

FIG. 7-3C depicts circuitry on an instrument that may be used to readsignals from a sensor comprising four active areas, according to someembodiments.

FIG. 7-4A depicts temporal emission characteristics for two differentemitters that may be used for sample analysis, according to someembodiments.

FIG. 7-4B depicts temporal evolution of an excitation source andluminescence from a sample, according to some embodiments.

FIG. 7-4C illustrates time-delay sampling, according to someembodiments.

FIG. 7-4D depicts temporal emission characteristics for two differentemitters, according to some embodiments.

FIG. 7-4E depicts voltage dynamics at a charge-accumulation node of asensor, according to some embodiments.

FIG. 7-4F depicts a double read of a sensor segment without reset,according to some embodiments.

FIG. 7-4G and FIG. 7-4H illustrate first and second read signal levelsassociated with two emitters having temporally-distinct emissioncharacteristics, according to some embodiments.

FIG. 7-5 illustrates a schematic diagram of a pixel with time resolutioncapability, according to some embodiments.

FIG. 7-6 illustrates a schematic diagram of a pixel with time resolutioncapability, according to some embodiments.

FIG. 8-1A and FIG. 8-1B depict spectral excitation bands of excitationsources, according to some embodiments.

FIG. 8-2A is a schematic of a coherent light source, according to someembodiments.

FIG. 8-2B illustrates temporal intensity profiles of an excitationsource, according to some embodiments.

FIG. 8-3 illustrates relaxation oscillations and a light signal from ofan excitation source, according to some embodiments.

FIG. 8-4 illustrates the use of a tailored electrical pulse to reducethe power of a tail in an output optical pulse, according to someembodiments.

FIG. 8-5 and FIG. 8-6 illustrates the optical output power of anexcitation source as a function of time, according to some embodiments.

FIG. 8-7 illustrates a higher current at higher frequencies resultingfrom using a greater number of wire bonds, according to someembodiments.

FIG. 8-8A is a schematic of a transmission line pulsar, according tosome embodiments.

FIG. 8-8B illustrates temporal profiles of light pulses obtained from atransmission line, according to some embodiments.

FIG. 8-9 and FIG. 8-10 are schematics of example circuits for generatingpulsed excitation light, according to some embodiments.

FIG. 8-11A illustrates an example circuit having one RF amplifier thatmay be used to produce a tailored electrical signal as an output pulse,according to some embodiments.

FIG. 8-11B illustrates an electrical pulse profile obtained from thecircuit of FIG. 8-11A.

FIG. 8-12A illustrates an example circuit having one RF amplifier thatmay be used to produce a tailored electrical signal as an output pulse,according to some embodiments.

FIG. 8-12B illustrates an electrical pulse profile obtained from thecircuit of FIG. 8-12A.

FIG. 8-13A illustrates a schematic for combining four different sources,according to some embodiments.

FIG. 8-13B illustrates the current, power efficiency, and voltage forfour sources as a function of impedance, according to some embodiments.

FIG. 9-1 depicts a method of operation of a compact apparatus that maybe used for rapid, mobile analysis of biological and chemical specimens,according to some embodiments.

FIG. 9-2 depicts a calibration procedure, according to some embodiments.

FIG. 9-3 depicts a data-analysis procedure, according to someembodiments.

FIG. 10-1 is a schematic of single molecule nucleic acid sequencing,according to some embodiments.

FIG. 10-2 schematically illustrates a sequencing process in a singlesample well over time.

FIG. 10-3 illustrates a process for preparing a sample well surface,according to some embodiments.

FIG. 10-4 illustrates example lifetime measurements, according to someembodiments.

FIG. 10-5 depicts a Fresnel lens integrated with a sensor, according tosome embodiments.

FIG. 10-6 illustrates the luminescent lifetimes of four markers,according to some embodiments.

FIG. 10-7 illustrates 16 time bins used by a sensor to distinguish themarkers from FIG. 10-6.

FIG. 10-8 illustrates the luminescent lifetimes of four markers,according to some embodiments.

FIG. 10-9 illustrates 13 time bins used by a sensor to distinguish themarkers from FIG. 10-6.

FIG. 10-10 illustrates a lifetime and spectral measurement scheme,according to some embodiments.

FIG. 10-11 illustrates the separation between four markers based onlifetime and emission wavelength, according to some embodiments.

FIG. 10-12 illustrates the emission spectra of three markers, accordingto some embodiments.

FIG. 10-13 illustrates the fluorescence lifetimes of four markers,according to some embodiments.

FIG. 10-14 illustrates signal profiles of the three markers from FIG.10-12 using a four-segment sensor, according to some embodiments.

FIG. 10-15 illustrates the fluorescence lifetimes of four markers,according to some embodiments.

FIG. 10-16 illustrates signal profiles of ATRho14 using a four-segmentsensor, according to some embodiments.

FIG. 10-17 illustrates a lifetime and absorption energy measurementscheme, according to some embodiments.

FIG. 10-18 illustrates the absorption spectra of four markers, accordingto some embodiments.

FIG. 10-19 illustrates the lifetime measurements of two markers,according to some embodiments.

FIG. 10-20 illustrates 8 time bins used by a sensor to distinguish themarkers from FIG. 10-19.

FIG. 10-21 illustrates the signal profiles of four markers, according tosome embodiments.

FIG. 10-22 illustrates the emission spectrum of four markers, accordingto some embodiments.

FIG. 10-23 depicts a computing environment, according to someembodiments.

FIG. 11-1 illustrates a method of fabricating a sample well, accordingto some embodiments.

FIG. 11-2 illustrates a method of fabricating a sample well using alift-off method, according to some embodiments.

FIG. 11-3 illustrates a method of forming a sample well, according tosome embodiments.

FIG. 11-4 illustrates a method of forming a concentric grating,according to some embodiments.

FIG. 11-5 illustrates a method of forming a concentric grating,according to some embodiments.

FIG. 11-6 illustrates a method of positioning the nanoaperture,according to some embodiments.

FIG. 11-7 illustrates a method of lens array fabrication, according tosome embodiments.

FIG. 11-8 illustrates a refractive lens array, according to someembodiments.

FIG. 11-9 illustrates a refractive lens array, according to someembodiments.

FIG. 11-10 illustrates a method of forming a lens, according to someembodiments.

FIG. 11-11 illustrates a method of forming a lens, according to someembodiments.

FIG. 11-12 illustrates a diffractive optical element, according to someembodiments.

FIG. 11-13 illustrates two unit cell layers used to form a diffractiveoptical element, according to some embodiments.

FIG. 11-14 illustrates a diffractive lens pattern according to someembodiments.

FIG. 11-15 and FIG. 11-16 illustrate a method of fabricating adiffractive optical element, according to some embodiments.

FIG. 11-17 and FIG. 11-18 and FIG. 11-19 illustrate a method offabricating an embedded Fresnel lens, according to some embodiments.

The features and advantages of embodiments of the present applicationwill become more apparent from the detailed description set forth belowwhen taken in conjunction with the drawings.

DETAILED DESCRIPTION

The inventors have recognized and appreciated that conventionalapparatuses for performing bioassays are large, expensive and requireadvanced laboratory techniques to perform. Many types of bioassaysdepend on the detection of single molecules in a specimen.Conventionally single molecule detection may require large, bulky lasersystems used to generate high intensity light needed for excitation ofmolecules. In addition, bulky optical components may be used to directthe laser light to the specimen and additional optical components may beused to direct luminescent light from the specimen to a sensor. Theseconventional optical components may require precise alignment andstabilization. The conventional laboratory equipment and trainingrequired to use this conventional equipment may result in complex,expensive bioassays.

The inventors have recognized and appreciated that there is a need for adevice that can simply and inexpensively analyze biological and/orchemical specimens to determine the identity of its constituent parts.An application of such a device may be for sequencing a biomolecule,such as a nucleic acid molecule or a polypeptide (e.g., protein) havinga plurality of amino acids. A compact, high-speed apparatus forperforming detection and quantitation of single molecules or particlescould reduce the cost of performing complex quantitative measurements ofbiological and/or chemical samples and rapidly advance the rate ofbiochemical technological discoveries. Moreover, a cost-effective devicethat is readily transportable could transform not only the way bioassaysare performed in the developed world, but provide people in developingregions, for the first time, ready access to essential diagnostic teststhat could dramatically improve their health and well-being. Forexample, in some embodiments, an apparatus for performing bioassays isused to perform diagnostic tests of biological samples, such as blood,urine and/or saliva. The apparatus may be used by individuals in theirhome, by a doctor in remote clinics of developing countries or any otherlocation, such as rural doctors' offices. Such diagnostic tests caninclude the detection of biomolecules in a biological sample of asubject, such as a nucleic acid molecule or protein. In some examples,diagnostic tests include sequencing a nucleic acid molecule in abiological sample of a subject, such as sequencing of cell freedeoxyribonucleic acid molecules or expression products in a biologicalsample of the subject.

The inventors have also recognized and appreciated that, when a sampleis tagged with a plurality of different types of luminescent markers,any suitable characteristic of luminescent markers may be used toidentify the type of marker that is present in a particular pixel of theintegrated device. For example, characteristics of the luminescenceemitted by the markers and/or characteristics of the excitationabsorption may be used to identify the markers. In some embodiments, theemission energy of the luminescence (which is directly related to thewavelength of the light) may be used to distinguish a first type ofmarker from a second type of marker. Additionally, or alternatively,luminescence lifetime measurements may also be used to identify the typeof marker present at a particular pixel. In some embodiments,luminescence lifetime measurements may be made with a pulsed excitationsource using a sensor capable of distinguishing a time when a photon isdetected with sufficient resolution to obtain lifetime information.Additionally, or alternatively, the energy of the excitation lightabsorbed by the different types of markers may be used to identify thetype of marker present at a particular pixel. For example, a firstmarker may absorb light of a first wavelength, but not equally absorblight of a second wavelength, while a second marker may absorb light ofthe second wavelength, but not equally absorb light of the firstwavelength. In this way, when more than one excitation light source,each with a different excitation energy, may be used to illuminate thesample in an interleaved manner, the absorption energy of the markerscan be used to identify which type of marker is present in a sample.Different markers may also have different luminescent intensities.Accordingly, the detected intensity of the luminescence may also be usedto identify the type of marker present at a particular pixel.

One non-limiting example of an application of a device contemplated bythe inventors is a device capable of performing sequencing of abiomolecule, such as a nucleic acid or a polypeptide (e.g. protein)having a plurality of amino acids. Diagnostic tests that may beperformed using such a device include sequencing a nucleic acid moleculein a biological sample of a subject, such as sequencing of cell freedeoxyribonucleic acid molecules or expression products in a biologicalsample of the subject.

The present application provides devices, systems and methods fordetecting biomolecules or subunits thereof, such as nucleic acidmolecules. Such detection can include sequencing. A biomolecule may beextracted from a biological sample obtained from a subject. Thebiological sample may be extracted from a bodily fluid or tissue of thesubject, such as breath, saliva, urine or blood (e.g., whole blood orplasma). The subject may be suspected of having a health condition, suchas a disease (e.g., cancer). In some examples, one or more nucleic acidmolecules are extracted from the bodily fluid or tissue of the subject.The one or more nucleic acids may be extracted from one or more cellsobtained from the subject, such as part of a tissue of the subject, orobtained from a cell-free bodily fluid of the subject, such as wholeblood.

Sequencing can include the determination of individual subunits of atemplate biomolecule (e.g., nucleic acid molecule) by synthesizinganother biomolecule that is complementary or analogous to the template,such as by synthesizing a nucleic acid molecule that is complementary toa template nucleic acid molecule and identifying the incorporation ofnucleotides with time (i.e., sequencing by synthesis). As analternative, sequencing can include the direct identification ofindividual subunits of the biomolecule.

During sequencing, signals indicative of individual subunits of abiomolecule may be collected in memory and processed in real time or ata later point in time to determine a sequence of the biomolecule. Suchprocessing can include a comparison of the signals to reference signalsthat enable the identification of the individual subunits, which in somecases yields reads. Reads may be sequences of sufficient length (e.g.,at least about 30 base pairs (bp)) that can be used to identify a largersequence or region, e.g., that can be aligned to a location on achromosome or genomic region or gene.

Individual subunits of biomolecules may be identified using markers. Insome examples, luminescent markers are used to identified individualsubunits of biomolecules. Some embodiments use luminescent markers (alsoreferred to herein as “markers”), which may be exogenous or endogenousmarkers. Exogenous markers may be external luminescent markers used as areporter and/or tag for luminescent labeling. Examples of exogenousmarkers may include, but are not limited to, fluorescent molecules,fluorophores, fluorescent dyes, fluorescent stains, organic dyes,fluorescent proteins, species that participate in fluorescence resonanceenergy transfer (FRET), enzymes, and/or quantum dots. Other exogenousmarkers are known in the art. Such exogenous markers may be conjugatedto a probe or functional group (e.g., molecule, ion, and/or ligand) thatspecifically binds to a particular target or component. Attaching anexogenous tag or reporter to a probe allows identification of the targetthrough detection of the presence of the exogenous tag or reporter.Examples of probes may include proteins, nucleic acid (e.g., DNA, RNA)molecules, lipids and antibody probes. The combination of an exogenousmarker and a functional group may form any suitable probes, tags, and/orlabels used for detection, including molecular probes, labeled probes,hybridization probes, antibody probes, protein probes (e.g.,biotin-binding probes), enzyme labels, fluorescent probes, fluorescenttags, and/or enzyme reporters.

Although the present disclosure makes reference to luminescent markers,other types of markers may be used with devices, systems and methodsprovided herein. Such markers may be mass tags, electrostatic tags, orelectrochemical labels.

While exogenous markers may be added to a sample, endogenous markers maybe already part of the sample. Endogenous markers may include anyluminescent marker present that may luminesce or “autofluoresce” in thepresence of excitation energy. Autofluorescence of endogenousfluorophores may provide for label-free and noninvasive labeling withoutrequiring the introduction of exogenous fluorophores. Examples of suchendogenous fluorophores may include hemoglobin, oxyhemoglobin, lipids,collagen and elastin crosslinks, reduced nicotinamide adeninedinucleotide (NADH), oxidized flavins (FAD and FMN), lipofuscin,keratin, and/or prophyrins, by way of example and not limitation.

While some embodiments may be directed to diagnostic testing bydetecting single molecules in a specimen, the inventors have alsorecognized that the single molecule detection capabilities of thepresent disclosure may be used to perform polypeptide (e.g., protein)sequencing or nucleic acid (e.g., DNA, RNA) sequencing of one or morenucleic acid segments of, for example, genes. Nucleic acid sequencingtechnologies may vary in the methods used to determine the nucleic acidsequence as well as in the rate, read length, and incidence of errors inthe sequencing process. For example, some nucleic acid sequencingmethods are based on sequencing by synthesis, in which the identity of anucleotide is determined as the nucleotide is incorporated into a newlysynthesized strand of nucleic acid that is complementary to the targetnucleic acid.

Having recognized the need for simple, less complex apparatuses forperforming single molecule detection and/or nucleic acid sequencing, theinventors have conceived of techniques for detecting single moleculesusing sets of luminescent tags to label different molecules using setsof tags, such as optical (e.g., luminescent) tags, to label differentmolecules. Such single molecules may be nucleotides or amino acidshaving tags. Tags may be detected while bound to single molecules, uponrelease from the single molecules, or while bound to and upon releasefrom the single molecules. In some examples, tags are luminescent tags.Each luminescent tag in a selected set is associated with a respectivemolecule. For example, a set of four tags may be used to “label” thenucleobases present in DNA—each tag of the set being associated with adifferent nucleobase, e.g., a first tag being associated with adenine(A), a second tag being associated with cytosine (C), a third tag beingassociated with guanine (G), and a fourth tag being associated withthymine (T). Moreover, each of the luminescent tags in the set of tagshas different properties that may be used to distinguish a first tag ofthe set from the other tags in the set. In this way, each tag isuniquely identifiable using one or more of these distinguishingcharacteristics. By way of example and not limitation, thecharacteristics of the tags that may be used to distinguish one tag fromanother may include the emission energy and/or wavelength of the lightthat is emitted by the tag in response to excitation energy and/or thewavelength of the excitation light that is absorbed by a particular tagto place the tag in an excited state.

Luminescent markers vary in the wavelength of light they emit, thetemporal characteristics of the light they emit (e.g., their emissiondecay time periods), and their response to excitation energy.Accordingly, luminescent markers may be identified or discriminated fromother luminescent markers based on detecting these properties. Suchidentification or discrimination techniques may be used alone or in anysuitable combination.

In some embodiments, an integrated photodetector as described in thepresent application can measure or discriminate luminance lifetimes,such as fluorescence lifetimes. Lifetime measurements are based onexciting one or more markers (e.g., fluorescent molecules), andmeasuring the time variation in the emitted luminescence. Theprobability of a marker to emit a photon after the marker reaches anexcited state decreases exponentially over time. The rate at which theprobability decreases may be characteristic of a marker, and may bedifferent for different markers. Detecting the temporal characteristicsof light emitted by markers may allow identifying markers and/ordiscriminating markers with respect to one another.

After reaching an excited state, a marker may emit a photon with acertain probability at a given time. The probability of a photon beingemitted from an excited marker may decrease over time after excitationof the marker. The decrease in the probability of a photon being emittedover time may be represented by an exponential decay functionp(t)=e{circumflex over ( )}(−t/τ), where p(t) is the probability ofphoton emission at a time, t, and τ is a temporal parameter of themarker. The temporal parameter τ indicates a time after excitation whenthe probability of the marker emitting a photon is a certain value. Thetemporal parameter, τ, is a property of a marker that may be distinctfrom its absorption and emission spectral properties. Such a temporalparameter, τ, is referred to as the luminance lifetime, the fluorescencelifetime or simply the “lifetime” of a marker.

FIG. 1-1 plots the probability of a photon being emitted as a functionof time for two markers with different lifetimes. The marker representedby probability curve B has a probability of emission that decays morequickly than the probability of emission for the marker represented byprobability curve A. The marker represented by probability curve B has ashorter temporal parameter, τ, or lifetime than the marker representedby probability curve A. Markers may have lifetimes ranging from 0.1-20ns, in some embodiments. However, the techniques described herein arenot limited as to the lifetimes of the marker(s) used.

The lifetime of a marker may be used to distinguish among more than onemarker, and/or may be used to identify marker(s). In some embodiments,lifetime measurements may be performed in which a plurality of markershaving different lifetimes is excited by an excitation source. As anexample, four markers having lifetimes of 0.5, 1, 2, and 3 nanoseconds,respectively, may be excited by a light source that emits light having aselected wavelength (e.g., 635 nm, by way of example). The markers maybe identified or differentiated from each other based on measuring thelifetime of the light emitted by the markers.

Lifetime measurements may use relative intensity measurements bycomparing how intensity changes over time, as opposed to absoluteintensity values. As a result, lifetime measurements may avoid some ofthe difficulties of absolute intensity measurements. Absolute intensitymeasurements may depend on the concentration of markers present andcalibration steps may be needed for varying marker concentrations. Bycontrast, lifetime measurements may be insensitive to the concentrationof markers.

Embodiments may use any suitable combination of tag characteristics todistinguish a first tag in a set of tags from the other tags in the sameset. For example, some embodiments may use only the timing informationof the emission light from the tags to identify the tags. In suchembodiments, each tag in a selected set of tags has a different emissionlifetime from the other tags in the set and the luminescent tags are allexcited by light from a single excitation source. FIG. 1-2A illustratesthe emission timing from four luminescent tags according to anembodiment where the four tags exhibit different average emissionlifetimes (τ). The probability that a tag is measured to have a lifetimeof a particular value is referred to herein as the tag's “emissiontiming.” A first emission timing 1-101 from a first luminescent tag hasa peak probability of having a lifetime of at τ1, a second emissiontiming 1-102 from a second luminescent tag has a peak probability ofhaving a lifetime of at τ2, a third emission timing 1-103 from a thirdluminescent tag has a peak probability of having a lifetime of at τ3,and a fourth emission timing 1-104 from a fourth luminescent tag has apeak probability of having a lifetime of at τ4. In this embodiment, thelifetime probability peaks of the four luminescent tags may have anysuitable values that satisfy the relation τ1<τ2<τ3<τ4. The four timingemission graphs may or may not overlap due to slight variations in thelifetime of a particular luminescent tag, as illustrated in FIG. 1-2A.In this embodiment, the excitation wavelength at which each of the fourtags maximally absorbs light from the excitation source is substantiallyequal, but that need not be the case. Using the above tag set, fourdifferent molecules may be labeled with a respective tag from the tagset, the tags may be excited using a single excitation source, and thetags can be distinguished from one another by detecting the emissionlifetime of the tags using an optical system and sensors. While FIG.1-2A illustrates four different tags, it should be appreciated that anysuitable number of tags may be used.

Other embodiments may use any suitable combination of tagcharacteristics to determine the identity of the tag within a set oftags. Examples of the tag characteristics that may be used include, butare not limited to excitation wavelength, emission wavelength, andemission lifetime. The combination of tag characteristics form a phasespace and each tag may be represented as a point within this phasespace. Tags within a set of tags should be selected such that the“distance” between each tag within the set is sufficiently large thatthe detection mechanism can distinguish each tag from the other tags inthe set. For example, in some embodiments a set of tags may be selectedwhere a subset of the tags have the same emission wavelength, but havedifferent emission lifetimes and/or different excitation wavelengths. Inother embodiments, a set of tags may be selected where a subset of thetags have the same emission lifetime, but have different emissionwavelengths and/or different excitation wavelengths. In otherembodiments, a set of tags may be selected where a subset of the tagshave the same excitation wavelength, but have different emissionwavelengths and/or different emission lifetimes.

By way of example and not limitation, FIG. 1-2B illustrates the emissionspectra from four luminescent tags according to an embodiment where twoof the tags have a first peak emission wavelength and the other two tagshave a second peak emission wavelength. A first emission spectrum 1-105from a first luminescent tag has a peak emission wavelength at λ1, asecond emission spectrum 1-106 from a second luminescent tag also has apeak emission wavelength at λ1, a third emission spectrum 1-107 from athird luminescent tag has a peak emission wavelength at λ2, and a fourthemission spectrum 1-108 from a fourth luminescent tag also has a peakemission wavelength at λ2. In this embodiment, the emission peaks of thefour luminescent tags may have any suitable values that satisfy therelation λ1<λ2. In embodiments such as this where the peak emissionwavelength is the same for more than one luminescent tag, a separatecharacteristic of the tags that have the same emission wavelength mustbe different. For example, the two tags that emit at λ1 may havedifferent emission lifetimes. FIG. 1-3A illustrates this situationschematically in a phase space spanned by the emission wavelength andthe emission lifetime. A first tag has an emission wavelength λ1 and aemission lifetime τ1, a second tag has an emission wavelength λ1 and aemission lifetime τ4, a third tag has an emission wavelength λ2 and aemission lifetime τ1, and a fourth tag has an emission wavelength λ2 anda emission lifetime τ4. In this way, all four tags in the tag set shownin FIG. 1-3A are distinguishable from one another. Using such a tag setallows distinguishing between four tags even when the absorptionwavelengths for the four dyes are identical. This is possible using asensor that can detect the time of emission of the photoluminescence aswell as the emission wavelength.

By way of example and not limitation, FIG. 1-2C illustrates theabsorption spectra from four luminescent tags according to anotherembodiment. In this embodiment, two of the tags have a first peakabsorption wavelength and the other two tags have a second peakabsorption wavelength. A first absorption spectrum 1-109 for the firstluminescent tag has a peak absorption wavelength at λ3, a secondabsorption spectrum 1-110 for the second luminescent tag has a peakabsorption wavelength at λ4, a third absorption spectrum 1-111 for thethird luminescent tag has a peak absorption wavelength at λ3, and afourth absorption spectrum 1-112 for the fourth luminescent tag has apeak absorption wavelength at λ4. Note that the tags that share anabsorption peak wavelength in FIG. 1-2C are distinguishable via anothertag characteristic, such as emission lifetime. FIG. 1-3B illustratesthis situation schematically in a phase space spanned by the absorptionwavelength and the emission lifetime. A first tag has an absorptionwavelength λ3 and an emission lifetime τ1, a second tag has anabsorption wavelength λ3 and an emission lifetime τ4, a third tag has anabsorption wavelength λ4 and a emission lifetime τ1, and a fourth taghas an absorption wavelength λ4 and a emission lifetime τ4. In this way,all four tags in the tag set shown in FIG. 1-3A are distinguishable fromone another.

Using such a tag set allows distinguishing between four tags even whenthe emission wavelengths for the four dyes are indistinguishable. Thisis possible using two excitation sources that emit at differentwavelengths or a single excitation source capable of emitting atmultiple wavelengths in connection with a sensor that can detect thetime of emission of the photoluminescence. If the wavelength of theexcitation light is known for each detected emission event, then it canbe determined which tag was present. The excitation source(s) mayalternate between a first excitation wavelength and a second excitationwavelength, which is referred to as interleaving. Alternatively, two ormore pulses of the first excitation wavelength may be used followed bytwo or more pulses of the second excitation wavelength.

The number of excitation sources or excitation wavelengths used todistinguish the tags is not limited to two, and in some embodiments morethan two excitation wavelengths or energies may be used to distinguishthe tags. In such embodiments, tags may be distinguished by theintensity or number of photons emitted in response to multipleexcitation wavelengths. A tag may be distinguishable from among multipletags by detecting the amount of photons emitted in response to exposingthe tag to a certain excitation wavelength. In some embodiments, a tagmay be distinguished by identifying the excitation energy from amongmultiple excitation energies where the tag emitted the highest number ofphotons. In other embodiments, the amount of emitted photons from a tagin response to different excitation energies may be used to identify thetag. A first tag that has a higher probability of emitting photons inresponse to a first excitation energy than a second excitation energymay be distinguished from a second tag that has a higher probability ofemitting photons in response to the second excitation energy than thefirst excitation energy. In this manner, tags having distinguishableprobabilities of emitting certain amounts of photons in response todifferent excitation energies may be identified by measuring the emittedphotons while exposing an unknown tag to the different excitationenergies. In such embodiments, a tag may be exposed to multipleexcitation energies and identification of the tag may be achieved bydetermining whether the tag emitted any light and/or an amount ofphotons emitted. Any suitable number of excitation energy sources may beused. In some embodiments, four different excitation energies may beused to distinguish among different tags (e.g., four different tags). Insome embodiments, three different excitation energies may be used todistinguish among different tags. Other characteristics of a tag may beused to distinguish the presence of a tag in combination with the amountof photons emitted in response to different excitation energies,including emission lifetime and emission spectra.

In other embodiments more than two characteristics of the tags in a tagset may be used to distinguish which tag is present. FIG. 1-4illustrates an illustrative phase space spanned by the absorptionwavelength, the emission wavelength and the emission lifetime of thetags. In FIG. 1-4, eight different tags are distributed in phase space.Four of the eight tags have the same emission wavelength, a differentfour tags have the same absorption wavelength and a different four tagshave the same emission lifetime. However, each of the tags isdistinguishable from every other tag when all three characteristics ofthe tags are considered. Embodiments are not limited to any number oftags. This concept can be extended to include any number of tags thatmay be distinguished from one another using at least these three tagcharacteristics.

While not illustrated in the figures, other embodiments may determinethe identity of a luminescent tag based on the absorption frequencyalone. Such embodiments are possible if the excitation light can betuned to specific wavelengths that match the absorption spectrum of thetags in a tag set. In such embodiments, the optical system and sensorused to direct and detect the light emitted from each tag does not needto be capable of detecting the wavelength of the emitted light. This maybe advantageous in some embodiments because it reduces the complexity ofthe optical system and sensors because detecting the emission wavelengthis not required in such embodiments.

As discussed above, the inventors have recognized and appreciated theneed for being able to distinguish different luminescent tags from oneanother using various characteristics of the tags. The type ofcharacteristics used to determine the identity of a tag impacts thephysical device used to perform this analysis. The present applicationdiscloses several embodiments of an apparatus, device, instrument andmethods for performing these different experiments.

Briefly, the inventors have recognized and appreciated that a pixelatedsensor device with a relatively large number of pixels (e.g., hundreds,thousands, millions or more) that allows for the detection of aplurality of individual molecules or particles in parallel. Such singlemolecules may be nucleotides or amino acids having tags. Tags may bedetected while bound to single molecules, upon release from the singlemolecules, or while bound to and upon release from the single molecules.In some examples, tags are luminescent tags. The molecules may be, byway of example and not limitation, proteins and/or nucleic acids (e.g.,DNA, RNA). Moreover, a high-speed device that can acquire data at morethan one hundred frames per second allows for the detection and analysisof dynamic processes or changes that occur over time within the samplebeing analyzed.

The inventors have recognized and appreciated that a low-cost,single-use disposable assay chip may be used in connection with aninstrument that includes an excitation light source, optics, and a lightsensor to measure an optical signal (e.g., luminescent light) emittedfrom biological samples. Using a low-cost assay chip reduces the cost ofperforming a given bioassay. A biological sample is placed onto theassay chip and, upon completion of a single bioassay, may be discarded.In some embodiments, more than one type of sample may be analyzedsimultaneously, in parallel, by placing multiple samples on differentportions of the assay chip at the same time. The assay chip interfaceswith the more expensive, multi-use instrument, which may be usedrepeatedly with many different disposable assay chips. A low-cost assaychip that interfaces with a compact, portable instrument may be usedanywhere in the world, without the constraint of high-cost biologicallaboratories requiring laboratory expertise to analyze samples. Thus,automated bioanalytics may be brought to regions of the world thatpreviously could not perform quantitative analysis of biologicalsamples. For example, blood tests for infants may be performed byplacing a blood sample on a disposable assay chip, placing thedisposable assay chip into the small, portable instrument for analysis,and processing the results by a computer that connects to the instrumentfor immediate review by a user. The data may also be transmitted over adata network to a remote location to be analyzed, and/or archived forsubsequent clinical analyses. Alternatively, the instrument may includeone or more processors for analyzing the data obtained from the sensorsof the instrument.

Various embodiments are described in more detail below.

I. OVERVIEW OF THE APPARATUS ACCORDING TO SOME EMBODIMENTS

A schematic overview of the apparatus 2-100 is illustrated in FIG. 2-1.The system comprises both an assay chip 2-110 and an instrument 2-120comprising an excitation source 2-121 and at least one sensor 2-122. Theassay chip 2-110 interfaces with the instrument 2-120 using any suitableassay chip interface. For example, the assay chip interface of theinstrument 2-120 may include a socket (not illustrated) for receivingthe assay chip 2-110 and holding it in precise optical alignment withthe excitation source 2-110 and the at least one sensor 2-122. Theexternal excitation source 2-121 in the instrument 2-120 is configuredto provide excitation energy to the assay chip 2-110 for the purpose ofexciting a sample in the sample well 2-111 of the assay chip 2-110. Insome embodiments, the assay chip 2-110 has multiple pixels, the samplewell 2-111 of each pixel configured to receive a sample used in ananalysis independent from the other pixels. Each pixel of the assay chip2-110 comprises a sample well 2-211 for receiving, retaining andanalyzing a sample from the specimen being analyzed. Such pixels may bereferred to as “passive source pixels” since the pixels receiveexcitation energy from an excitation source separate from the pixel. Insome embodiments, there is a pixel in the instrument 2-120 correspondingto each pixel present on the assay chip 2-110. Each pixel of theinstrument 2-120 comprises at least one sensor for detecting emissionenergy emitted by the sample in response to the sample being illuminatedwith excitation energy from the excitation source 2-121. In someembodiments, each sensor may include multiple sub-sensors, eachsub-sensor configured to detect a different wavelength of emissionenergy from the sample. While more than one sub-sensor may detectemission energy of a certain wavelength, each sub-sensor may detect adifferent wavelength band of emission energy.

In some embodiments, optical elements for guiding and couplingexcitation energy from the excitation source 2-121 to the sample well2-111 are located both on the assay chip 2-110 and the instrument 2-120,as represented by arrow 2-101 in FIG. 2-1. Such source-to-well elementsmay include mirrors, lenses, dielectric coatings and beam combinerslocated on the instrument 2-120 to couple excitation energy to the assaychip 2-110 and lenses, plasmonic elements and dielectric coatings on theassay chip 1-110 to direct the excitation energy received from theinstrument 2-120 to the sample well 2-111. Additionally, in someembodiments, optical elements for guiding emission energy from thesample well 2-111 to the sensor 2-122 are located on the assay chip2-110 and the instrument 2-120, as represented by arrow 2-102 in FIG.2-1. Such well-to-sample elements may include may include lenses,plasmonic elements and dielectric coatings located on the assay chip2-110 to direct emission energy from the assay chip 2-110 to theinstrument 2-120 and lenses, mirrors, dielectric coatings, filters anddiffractive optics on the instrument 1-120 to direct the emission energyreceived from the assay chip 2-110 to the sensor 2-111. In someembodiments, a single component may play a role both in couplingexcitation energy to a sample well and delivering emission energy fromthe sample well to the sensor.

In some embodiments, the assay chip 2-110 comprises a plurality ofpixels, each pixel associated with its own individual sample well 2-111and its own associated sensor 2-122 on the instrument 2-120. Theplurality of pixels may be arranged in an array and may have anysuitable number of pixels. For example, the assay chip may includeapproximately 1,000 pixels, 10,000 pixels, approximately 100,000 pixels,approximately 1,000,000 pixels, approximately 10,000,000 pixels, orapproximately 100,000,000 pixels.

In some embodiments, the instrument 2-120 includes a sensor chipcomprising a plurality of sensors 2-122 arranged as a plurality ofpixels. Each pixel of the sensor chip corresponds to a pixel in theassay chip 2-110. The plurality of pixels may be arranged in an arrayand may have any suitable number of pixels. In some embodiments, thesensor chip has the same number of pixels as the assay chip 2-110. Forexample, the sensor chip may include approximately 10,000 pixels,approximately 100,000 pixels, approximately 1,000,000 pixels,approximately 10,000,000 pixels, or approximately 100,000,000 pixels.

The instrument 2-120 interfaces with the assay chip 2-110 through anassay chip interface (not shown). The assay chip interface may includecomponents to position and/or align the assay chip 2-110 to theinstrument 2-120 to improve coupling of the excitation energy from theexcitation source 2-121 to the assay chip 2-110. In some embodiments,excitation source 2-121 includes multiple excitation sources that arecombined to deliver excitation energy to the assay chip 2-110. Themultiple excitation sources may be configured to produce multipleexcitation energies, corresponding to light of different wavelengths.

The instrument 2-120 includes a user interface 2-125 for controlling theoperation of the instrument. The user interface 2-125 is configured toallow a user to input information into the instrument, such as commandsand/or settings used to control the functioning of the instrument. Insome embodiments, the user interface 2-125 may include buttons,switches, dials, and a microphone for voice commands. Additionally, theuser interface 2-125 may allow a user to receive feedback on theperformance of the instrument and/or assay chip, such as properalignment and/or information obtained by readout signals from thesensors on the sensor chip. In some embodiments, the user interface2-125 may provide feedback using a speaker to provide audible feedback,and indicator lights and/or a display screen for providing visualfeedback. In some embodiments, the instrument 2-120 includes a computerinterface 2-124 used to connect with a computing device 2-130. Anysuitable computer interface 2-124 and computing device 2-130 may beused. For example, the computer interface 2-124 may be a USB interfaceor a firewire interface. The computing device 2-130 may be any generalpurpose computer, such as a laptop, desktop, or tablet computer, or amobile device such as a cellular telephone. The computer interface 2-124facilitates communication of information between the instrument 2-120and the computing device 2-130. Input information for controlling and/orconfiguring the instrument 2-120 may be provided through the computingdevice 2-130 connected to the computer interface 2-124 of theinstrument. Additionally, output information may be received by thecomputing device 2-130 through the computer interface 2-124. Such outputinformation may include feedback about performance of the instrument2-120 and information from the readout signals of the sensor 2-122. Theinstrument 2-120 may also include a processing device 2-123 foranalyzing data received from the sensor 2-122. In some embodiments, theprocessing device 2-123 may be a general purpose processor (e.g., acentral processing unit (CPU), a field programmable gate array (FPGA) ora custom integrated circuit, such as an application specific integratedcircuit (ASIC). In some embodiments, the processing of data from thesensor 1-122 may be performed by both the processing device 2-123 andthe external computing device 2-130. In other embodiments, the computingdevice 2-130 may be omitted and processing of data from the sensor 2-122may be performed solely by processing device 2-123.

When the excitation source 2-121 illuminates the assay chip 2-110 withexcitation energy, samples within one or more pixels of the assay chip2-110 may be excited. In some embodiments, a specimen is labeled withmultiple markers and the multiple markers, each associated with adifferent sample within the specimen, are identifiable by the emissionenergy. The path from the sample well 2-111 to the sensor 2-122 mayinclude one or more components that aid in identifying the multiplemarkers based on emission energy. Components may focus emission energytowards the sensor 2-122 and may additionally or alternatively spatiallyseparate emission energies that have different characteristic energies,and therefore different wavelengths. In some embodiments, the assay chip2-110 may include components that direct emission energy towards thesensor 2-122 and the instrument 2-120 may include components forspatially separating emission energy of different wavelengths. Forexample, optical filters or diffractive optics may be used to couple thewavelength of the emission energy to a spatial degree of freedom. Thesensor or sensor region may contain multiple sub-sensors configured todetect a spatial distribution of the emission energy that depends on theradiation pattern. Luminescent tags that emit different emissionenergies and/or spectral ranges may form different radiation patterns.The sensor or sensor region may detect information about the spatialdistribution of the emission energy that can be used to identify amarker among the multiple markers.

The emission energy from the sample in the sample well 2-110 may bedetected by the sensor 2-122 and converted to at least one electricalsignal. The electrical signals may be transmitted along conducting linesin the circuitry of the instrument 2-120 and processed and/or analyzedby the processing device 2-123 and/or computing device 2-130.

FIG. 2-2 is a top view of the assay chip 2-110 and the top view of thesensor chip 2-260 and illustrates the correspondence between the pixelsof the two chips. The assay chip 2-110 comprises a plurality of pixels,each pixel including a sample well 2-111 formed in layer 2-221. Layer2-221 may comprise a conductive material including a metal, a highlydegeneratively-doped semiconductor, and graphene. In some embodiments,layer 2-221 may comprise a plurality of layers formed from one or moredifferent types of materials (e.g., metal, semiconductor, dielectric,insulator). The sensor chip 2-260 also comprises a plurality of pixels,each pixel including a sensor 2-121 formed in or on a substrate 2-247.The arrows in FIG. 2-2 illustrate the correspondence between two of thepixels of the assay chip 2-110 and two of the pixels of the sensor chip2-260. While not illustrated for the sake of clarity, each pixel of theassay chip 2-110 is associated with a pixel of the sensor chip 2-260.

Pixels of the assay chip 2-110 and the sensor chip 2-260 may have anysuitable size, shape, and arrangement. In some embodiments, pixels ofthe assay chip 2-100 and the sensor chip 2-260 may be arranged in arectangular or square configuration, as shown in FIG. 2-2.

An overview of some components associated with a single pixel of theassay chip 2-110 and a single pixel of the sensor chip 2-260 isillustrated in FIG. 2-3. The apparatus 2-100 comprises both the assaychip 2-110 and the instrument 2-120. In some embodiments, the assay chip2-110 is a disposable chip designed for the analysis of a singlespecimen. The assay chip 2-110 includes one or more metal layers 2-221,one or more dielectric layers 2-225 and focusing elements 2-227. In someembodiments, metal layer 2-221 includes a stack of layers, some of whichmay include absorbing layers. The instrument 2-120 includes one or moreexcitation sources 2-250, at least one polychroic mirror 2-230, and thesensor chip 2-260, which may include filtering elements 2-241, spectralsorting elements 2-243, focusing elements 2-245 and at least one sensor2-122 in or on the substrate 2-247. While FIG. 2-3 illustrates only asingle pixel of the assay chip 2-110 and only a single pixel of thesensor chip 2-260, some components of the instrument 2-120, such as theexcitation source 2-250, the polychroic mirror 2-230 and filteringelements 2-241, may be common to a plurality of the pixels. For example,in some embodiments, a single excitation source 2-250 and polychroicmirror 2-230 may direct the excitation energy to every pixel of theassay chip 2-110.

The sample well 2-211 within the metal layer 2-221 forms a sample volumefor a sample from the specimen to enter. In some embodiments, thespecimen may include bodily fluid, such as blood, urine or saliva. Theopenings at the end of the sample well 2-211 may be referred to as ananoaperture. The nanoaperture may have a width that is smaller than thewavelength of the excitation energy 2-251 emitted by excitation source2-250. A portion of the specimen, referred to as a sample, may enter thesample volume defined by the sample well 2-211. The sample may be anyparticle, molecule, protein, genetic material or any other samplepresent in the specimen.

Excitation source 2-250 emits excitation energy 2-251, which is directedtowards the sample well 2-211 to illuminate the sample. In someembodiments, excitation source 2-251 may be a single light source thatprovides excitation energy for all the pixels of assay chip 2-110. Thepolychroic mirror 2-230 reflects light from the excitation source 2-250and directs the excitation energy 2-251 towards one or more sample wells2-211 of the assay chip 2-110. Thus, in some embodiments, there may beonly a single polychroic mirror 2-230 that directs the excitation energytowards all the sample wells, rather than each pixel being associatedwith its own polychroic mirror. Similarly, there may be a one-to-manyrelationship between other optical elements used to direct theexcitation energy towards sample wells 2-211.

A concentric circular grating 2-223 may be formed adjacent to the bottomnanoaperture of the sample well 2-211. The concentric circular gratings2-223 may protrude from a bottom surface of the metal layer 2-221. Thesample well 2-211 may be located at or near the center of the circulargrating 2-223. Both the sub-wavelength scale of the nanoaperture of thesample well 2-211 and the concentric circular gratings 2-223 create afield enhancement effect that increases the intensity of the excitationenergy in the sample well 2-211, resulting in increased coupling of theexcitation energy to a sample present in the sample well 2-211. At leastsome of the time, the sample absorbs a photon from the excitation energyand emits a photon (referred to as “emission energy” 2-253) with anenergy less than that of the excitation energy 2-251. The emissionenergy 2-253 may be emitted in a downward direction. The circulargratings 2-223 act as plasmonic elements which may be used to decreasethe spread of the emission energy 2-253 and direct the emission energy2-253 towards an associated sensor.

The emission energy 2-253 travels through the dielectric layer 2-225,which may be a spacer layer used to allow the emission energy 2-253 topropagate some distance. The dielectric layer 2-225 may also providestructural strength to the assay chip 2-110. The emission energy 2-253then travels through one or more focusing elements 2-227 used to furtherdirect the emission energy 2-253 to the sensor 2-122 in the associatedpixel of the sensor chip 2-2260 within the instrument 2-120.

The polychroic mirror 2-230 may transmit the emission energy 2-253and/or reflect a portion of any excitation energy 2-251 reflected fromthe assay chip 2-110. The portion of the excitation light that is notreflected by the assay chip 2-110 is either transmitted through theassay chip or absorbed by the assay chip. To further reduce the amountof excitation energy 2-251 reflected by the assay chip 2-110 and notreflected by the polychroic mirror 2-230, filtering elements 2-241 maybe disposed in the optical path towards the sensor chip 2-260. Thefiltering components 2-241 may act to reduce excitation energy 2-251detected by sensor 2-122. The filtering elements 2-241 may include, byway of example and not limitation, a broadband filter, a notch filter oran edge filter, which transmit emission energy 2-253 but absorb and/orreflect excitation energy 2-251.

In some embodiments, to facilitate using spectral properties of theemission energy 2-253 to determine the identity of the marker in thesample well 2-211, spectral sorting elements 2-243 may be included onthe sensor chip 2-260 to couple the spectral degree of freedom of theemission energy 2-253 to the direction the emission energy 2-253 istraveling. For example, a diffractive optical element may be used todirect emission energy 2-253 of a first wavelength in a first directionand emission energy 2-253 of a second wavelength in a second direction.One or more focusing elements 2-245 may be used to direct the spectrallysorted light onto the sensor 2-122. The sensor 2-122 may include one ormore sub-sensors (not shown), each of which is associated with adifferent wavelength of the emission energy 2-253 based on theredirection of light of different wavelengths by the spectral sortingelement 2-243. In this manner, the one or more focusing elements 2-245may form different distribution patterns for emission energy havingdifferent characteristic wavelengths, which may allow for identificationof markers based on characteristic wavelength of emission energy.

In embodiments where lifetime of the emission energy 2-253 is used todetermine the identity of the marker in sample well 2-211, sensor 2-122may be capable of detecting when a photon of emission energy is absorbedby sensor 2-122. The sensor 2-122 may, for example, be a CMOS devicecapable of sorting detected photons into a plurality of time bins. Thelifetime may be determined by detecting a plurality of emission energyphotons resulting from a plurality of excitation pulses.

The above description of FIG. 2-3 is an overview of some, but notnecessarily all, of the components of the apparatus according to someembodiments. In some embodiments, one or more elements of FIG. 2-3 maybe absent or in a different location. The components of the assay chip2-210 and instrument 2-220 are described in more detail below.

The assay chip 2-110 and the instrument 2-120 may be mechanicallyaligned, detachably coupled and separable from one another. Theinstrument 2-120 may include an instrument housing, inside which amounting board is disposed. FIG. 2-4 illustrates at least some of thecomponents that may be included on the mounting board 2-405 of theinstrument 2-120. The mounting board 2-405, which may include a printedcircuit board, may have the sensor chip 2-260 (not visible in FIG. 2-4),a heat sink 2-407 and an optical housing 2-401 mounted thereto. Thevarious optical components of the instrument 2-120 may be disposedwithin the optical housing 2-401. In some embodiments, instrumenthousing and mounting board may have any suitable size and shape. Forexample, the mounting board may be substantially circular with adiameter of approximately 7-8″. The assay chip 2-110 couples to theoptical housing 2-401 to ensure alignment with the optical componentswithin the optical housing 2-401.

A chip holder frame 3-102 may be aligned with an opening of the opticalhousing 2-401. The assay chip 2-110 may be housed in chip holder frame2-401. In some embodiments, assay chip 2-110 may be situated on theunderside of chip holder frame 3-102 such that positioning of the chipholder frame 3-102 relative to instrument 2-120 locates assay chip 2-110on a side of chip holder frame 3-102 proximate to instrument 2-120. Thechip holder frame 3-102 may comprise any suitable material. In someembodiments, chip holder frame 3-102 may include a ferromagnetic metal(e.g., steel) such that chip holder frame 3-102 may align with anopening of optical housing 2-401 by one or more magnetic componentspositioned on a surface of the optical housing 2-401 that act to holdchip holder frame 3-102 in place.

In some embodiments, the assay chip 2-110 may be detachably coupled tothe instrument 2-120. One or more magnetic components 2-403 a, 2-403 b,and 2-403 c of any suitable shape and size, such as magnetic cylindersas shown in FIG. 2-4, may be placed around an opening of the opticalhousing 3-401 through which excitation energy exits the optical housing2-401. Additionally, the magnetic components 2-403 a, 2-403 b, and2-2-403 c may be calibrated such that the chip holder frame 3-102 isheld in alignment with the opening. The chip holder frame 3-102 may bepositioned with a micron-level accuracy using the magnetic components2-403 a, 2-403 b, and 2-2-403 c. In some embodiments, magneticcomponents 2-403 a, 2-403 b, and 2-2-403 c are used to create chipholder frame alignment. However, embodiments are not so limited and anysuitable number of magnetic, spring-loaded, pneumatic or other suchcomponents may be used to hold the chip in place in an alignedconfiguration. For example, the chip holder frame 3-102 may be held inplace with a non-magnetic element, such as a spring, air pressure, orsuction from a vacuum. Optionally, the chip holder frame 3-102 may beconstructed using any stiff material suitable for positioning the chipin alignment with the optical block.

According to some aspects of the present application, when the assaychip 2-110 is connected to the instrument 2-120, the distance betweenthe sample wells of the assay chip 2-110 and the sensors of the sensorchip 2-260 in instrument 2-120 may be within a desired distance toachieve a sufficient level of performance for the system. In someembodiments, the optical distance between the sample wells and thesensors may be less than 30 cm, less than 10 cm, less than 5 cm, or lessthan 1 cm.

II. ASSAY CHIP

In some embodiments, the assay chip 2-110 does not include any activeelectronic components. Both the excitation source 2-250 and the sensor2-122 for each pixel are located off-chip in the instrument 2-120.

In some embodiments, the assay chip 2-110 may be housed in a chip holderframe 3-102 as illustrated in FIG. 3-1A. The chip holder frame 3-102 maybe disposable and may be disposed of along with the assay chip 2-110after a single use. The assay chip 2-110 may be situated on theunderside of the chip holder frame 3-102, as illustrated in FIG. 3-1B.The chip holder frame 3-102 may comprise any suitable ferromagneticmetal, such as steel, such that the magnetic components 2-403 a, 2-403b, and 2-403 c fixed to the optical housing 2-401 hold the chip holderframe 3-102, and thus the assay chip 2-110, in place. In someembodiments, the chip holder frame 3-102 may be attached to the topsurface of the optical housing 2-401 as illustrated in FIG. 2-4.

In other embodiments, illustrated in FIG. 3-1C, the assay chip 2-110 maybe attached to a top surface of the chip holder frame 3-102. A plasticcap 3-103 surrounds the assay chip 2-110 such that the pixel array ofthe assay chip 2-110 is exposed via an opening in the plastic cap 3-103.A user of the assay chip 2-110 may place a specimen into the opening ofthe plastic cap 3-103. By being in contact with the top surface of theassay chip 2-110, the samples within the specimen may be introduced toone or more of the plurality of pixels of the assay chip 2-110 foranalysis. In some embodiments, no fluidic channels or device fordelivering portions of the sample to the pixels via forced fluid floware necessary.

In some embodiments, the assay chip may include layers of componentsstacked vertically. These components may include optical, electrical,chemical, biochemical, and structural elements. In some embodiments,each layer of the assay chip is the same for each pixel. What follows isa description of a single pixel, but every pixel in the array of pixelson an assay chip may have exactly same layout according to someembodiments.

A. Sample Well Layer

As illustrated in FIG. 2-3, and in more detail at FIG. 3-2, someembodiments include a sample well 2-211 formed at one or more pixels ofthe assay chip 2-110. A sample well may comprise a small volume orregion formed within metal layer 2-221 and arranged such that samplesmay diffuse into and out of the sample well from a specimen deposited onthe surface of the assay chip 2-110. In various embodiments, a samplewell 2-211 may be arranged to receive excitation energy from anexcitation source 2-250. Samples that diffuse into the sample well maybe retained, temporarily or permanently, within an excitation region3-215 of the sample well by an adherent 3-211. In the excitation region,a sample may be excited by excitation energy (e.g., excitation light3-245), and subsequently emit energy that may be observed and evaluatedto characterize the sample.

In further detail of operation, at least one sample 3-101 to be analyzedmay be introduced into a sample well 2-211, e.g., from a specimen (notshown) containing a fluid suspension of samples. Excitation energy 3-245from an excitation source 2-250 in the instrument 2-120 may excite thesample or at least one tag (also referred to as a biological marker,reporter, or probe) attached to the sample or otherwise associated withthe sample while it is within an excitation region 3-215 within thesample well. According to some embodiments, a tag may be a luminescentmolecule (e.g., a luminescent tag or probe) or quantum dot. In someimplementations, there may be more than one tag that is used to analyzea sample (e.g., distinct tags that are used for single-molecule geneticsequencing as described in “Real-Time DNA Sequencing from SinglePolymerase Molecules,” by J. Eid, et al., Science 323, p. 133 (2009),which is incorporated by reference). During and/or after excitation, thesample or tag may emit emission energy. When multiple tags are used,they may emit at different characteristic energies (and therefore havedifferent wavelengths) and/or emit with different temporalcharacteristics. In some embodiments, emission energy may include anynumber of wavelengths, for example, two, three, four, five, six, seven,or eight different wavelengths. The emissions from the sample well 2-211may radiate to a sensor 2-122 on the instrument 2-120 where they aredetected and converted into electrical signals that can be used tocharacterize the sample.

According to some embodiments, a sample well 2-211 may be a partiallyenclosed structure, as depicted in FIG. 3-2. In some implementations, asample well 2-211 comprises a sub-micron-sized hole or opening(characterized by at least one transverse dimension D_(sw)) formed in atleast one layer of material 2-221. The transverse dimension of thesample well may be between approximately 20 nanometers and approximately1 micron, according to some embodiments, though larger and smaller sizesmay be used in some implementations. A volume of the sample well 2-211may be between about 10⁻²¹ liters and about 10⁻¹⁵ liters, in someimplementations. A sample well may be formed as a waveguide that may, ormay not, support a propagating mode. In some embodiments, a sample wellmay be formed as a zero-mode waveguide (ZMW) having a cylindrical shape(or similar shape) with a diameter (or largest transverse dimension)D_(sw). A ZMW may be formed in a single metal layer as a nano-scale holethat does not support a propagating optical mode through the hole.

Because the sample well 2-211 has a small volume, detection ofsingle-sample events (e.g., single-molecule events) at each pixel may bepossible even though samples may be concentrated in an examined specimenat concentrations that are similar to those found in naturalenvironments. For example, micromolar concentrations of the sample maybe present in a specimen that is placed in contact with the assay chip,but at the pixel level only about. Sample wells of the assay 2-110 aresized such that, statistically, they most likely contain no sample orone sample, so that single molecule analysis may be performed. Forexample, in some embodiments 30-40% of the sample wells contain a singlesample. However, sample wells may contain more than one sample. Becausesingle-molecule or single-sample events may be analyzed at each pixel,the assay chip makes it possible to detect rare events that mayotherwise go unnoticed in ensemble averaged measurements.

A transverse dimension D_(sw) of a sample well may be between about 500nanometers (nm) and about one micron in some embodiments, between about250 nm and about 500 nm in some embodiments, between about 100 nm andabout 250 nm in some embodiments, and yet between about 20 nm and about100 nm in some embodiments. The transverse dimension of a sample wellmay be approximately 100 nm, approximately 130 nm, or approximately 190nm. According to some implementations, a transverse dimension of asample well is between approximately 80 nm and approximately 180 nm, orbetween approximately one-quarter and one-eighth of the excitationwavelength or emission wavelength. According to other implementations, atransverse dimension of a sample well is between approximately 120 nmand approximately 170 nm. In some embodiments, the depth or height ofthe sample well 2-211 may be between about 50 nm and about 500 nm. Insome implementations, the depth or height of the sample well 2-211 maybe between about 80 nm and about 200 nm. The layer of material 2-221that forms the sample well 2-211 may have a thickness or height orapproximately 50 nm, approximately 100 nm, approximately 150 nm,approximately 200 nm, or approximately 250 nm.

A sample well 2-211 having a sub-wavelength, transverse dimension canimprove operation of a pixel 2-100 of an assay chip 2-110 in at leasttwo ways. For example, excitation energy 3-245 incident on the samplewell from a side opposite the specimen may couple into the excitationregion 3-215 with exponentially decreasing power, and not propagatethrough the sample well to the specimen. As a result, excitation energyis increased in the excitation region where it excites a sample ofinterest, and is reduced in the specimen where it could excite othersamples that would contribute to background noise. Also, emission from asample retained at a base of the well is preferably directed toward thesensor on the instrument 2-120, since emission cannot propagate upthrough the sample well. Both of these effects can improvesignal-to-noise ratio at the pixel. The inventors have recognizedseveral aspects of the sample well that can be improved to further boostsignal-to-noise levels at the pixel. These aspects relate to well shapeand structure, and placement relative to adjacent optical and plasmonicstructures (described below) that aid in coupling excitation energy tothe sample well and emitted energy from the sample well.

According to some embodiments, a sample well 2-211 may be formed as asub-cutoff nano-aperture (SCN), which does not support a propagatingmode. For example, the sample well 2-211 may comprise acylindrically-shaped hole or bore in a conductive layer 2-221. Thecross-section of a sample well need not be round, and may be elliptical,square, rectangular, or polygonal in some embodiments. Excitation energy3-245 (e.g., visible or near infrared radiation) may enter the samplewell through an entrance aperture 3-212 that may be defined by walls3-214 of the sample well 2-211 at a first end of the well, as depictedin FIG. 3-2. When formed as an SCN, the excitation energy 3-245 maydecay exponentially along the SCN. In some implementations, thewaveguide may comprise an SCN for emitted energy from the sample, butmay not be an SCN for excitation energy. For example, the aperture andwaveguide formed by the sample well may be large enough to support apropagating mode for the excitation energy, since it may have a shorterwavelength than the emitted energy. The emission, at a longerwavelength, may be beyond a cut-off wavelength for a propagating mode inthe waveguide. According to some embodiments, the sample well 2-211 maycomprise an SCN for the excitation energy 3-245, such that the greatestintensity of excitation energy is localized to an excitation region3-215 of the sample well at an entrance to the sample well 2-211 (e.g.,localized near the interface between layer 3-235 and layer 2-221 asdepicted in FIG. 3-2). Such localization of the excitation energy canincrease the emission energy density from the sample, and furtherconfine the excitation energy near the entrance aperture 3-212, therebylimiting the observed emission to a single sample (e.g., a singlemolecule).

An example of excitation localization near an entrance of a sample wellthat comprises an SCN is depicted in FIG. 3-3. A numerical simulationwas carried out to determine intensity of excitation energy within andnear a sample well 2-211 formed as an SCN. The results show that theintensity of the excitation energy is about 70% of the incident energyat an entrance aperture of the sample well and drops to about 20% of theincident intensity within about 100 nm in the sample well. For thissimulation, the characteristic wavelength of the excitation energy was633 nm and the diameter of the sample well 2-211 was 140 nm. The samplewell 2-211 was formed in a layer of gold metal. Each horizontal divisionin the graph is 50 nm. As shown by the graph, more than one-half of theexcitation energy received in the sample well is localized to about 50nm within the entrance aperture 3-212 of the sample well 2-211.

To improve the intensity of excitation energy that is localized at thesample well 2-211, other sample well structures were developed andstudied by the inventors. FIG. 3-4 depicts an embodiment of a samplewell that includes a cavity or divot 3-216 at an excitation end of thesample well 2-211. As can be seen in the simulation results of FIG. 3-3,a region of higher excitation intensity exists just before the entranceaperture 2-212 of the sample well. Adding a divot 3-216 to the samplewell 2-211 allows a sample to move into a region of higher excitationintensity, according to some embodiments. In some implementations, theshape and structure of the divot alters the local excitation field(e.g., because of a difference in refractive index between the layer3-235 and fluid of the specimen in the sample well), and can furtherincrease the intensity of the excitation energy in the divot.

The divot may have any suitable shape. The divot may have a transverseshape that is substantially equivalent to a transverse shape of thesample well, e.g., round, elliptical, square, rectangular, polygonal,etc. In some embodiments, the sidewalls of the divot may besubstantially straight and vertical, like the walls of the sample well.In some implementations, the sidewalls of the divot may be sloped and/orcurved, as depicted in the drawing. The transverse dimension of thedivot may be approximately the same size as the transverse dimension ofthe sample well in some embodiments, may be smaller than the transversedimension of the sample well in some embodiments, or may be larger thanthe transverse dimension of the sample well in some embodiments. Thedivot 3-216 may extend between approximately 10 nm and approximately 200nm beyond the metallic layer 2-221 of the sample well. In someimplementations, the divot may extend between approximately 50 nm andapproximately 150 nm beyond the metallic layer 2-221 of the sample well.By forming the divot, the excitation region 3-215 may extend outside themetallic layer 2-221 of the sample well, as depicted in FIG. 3-4.

FIG. 3-5 depicts improvement of excitation energy at the excitationregion for a sample well containing a divot (shown in the leftsimulation image). For comparison, the excitation field is alsosimulated for a sample well without a divot, shown on the right. Thefield magnitude has been converted from a color rendering in theseplots, and the dark region at the base of the divot represents higherintensity than the light region within the sample well. The dark regionsabove the sample well represents the lowest intensity. As can be seen,the divot allows a sample 3-101 to move to a region of higher excitationintensity, and the divot also increases the localization of region ofhighest intensity at an excitation end of the sample well. Note that theregion of high intensity is more distributed for the sample well withoutthe divot. In some embodiments, the divot 3-216 provides an increase inexcitation energy at the excitation region by a factor of two or more.In some implementations, an increase of more than a factor of two can beobtained depending on the shape and depth of the divot. In thesesimulations, the sample well comprises a layer of Al that is 100 nmthick, with a divot that is 50 nm deep, with excitation energy at 635 nmwavelength.

FIG. 3-6 depicts another embodiment of a sample well 2-211 in which thesample well, including the divot 3-216, are formed over a protrusion3-615 at a surface of a substrate. A resulting structure for the samplewell may increase the excitation energy at the sample by more than afactor of two compared to a sample well shown in FIG. 3-2, and maydirect emission from the sample well toward the sensor in the instrument2-120. According to some embodiments, a protrusion 3-615 is patterned ina first layer 3-610 of material. The protrusion may be formed as acircular pedestal or a ridge with rectangular cross-section in someimplementations, and a second layer 3-620 of material may be depositedover the first layer and the protrusion. At the protrusion, the secondlayer may form a shape above the protrusion that approximates acylindrical portion 3-625, as depicted. In some embodiments, aconductive layer 3-230 (e.g., a reflective metal) may be deposited overthe second layer 3-620 and patterned to form a sample well 3-210 in theconductive layer above the protrusion. A divot 3-216 may then be etchedinto the second layer. The divot may extend between about 50 nm andabout 150 nm below the conductive layer 3-230. According to someembodiments, the first layer 3-610 and second layer 3-620 may beoptically transparent, and may or may not be formed of a same material.In some implementations, the first layer 3-610 may be formed from anoxide (e.g., SiO₂) or a nitride (e.g., Si₃N₄), and the second layer3-620 may be formed from an oxide or a nitride.

According to some embodiments, the conductive layer 3-230 above theprotrusion 3-625 is shaped approximately as a spherical reflector 3-630.The shape of the spherical portion may be controlled by selection of theprotrusion height h, diameter or transverse dimension w of theprotrusion, and a thickness t of the second layer 3-620. The location ofthe excitation region and position of the sample can be adjusted withrespect to an optical focal point of the cylindrical reflector byselection of the divot depth d. It may be appreciated that the sphericalreflector 3-630 can concentrate excitation energy at the excitationregion 3-215, and can also collect energy emitted from a sample andreflect and concentrate the radiation toward the sensor 3-260.

As noted above, a sample well may be formed in any suitable shape, andis not limited to only cylindrical shapes. In some implementations, asample well may be conic, tetrahedron, pentahedron, etc. FIG. 3-7Athrough FIG. 3-7F illustrates some example sample well shapes andstructures that may be used in some embodiments. A sample well 2-211 maybe formed to have a first aperture 3-212 that is larger than a secondaperture 3-218 for the excitation energy, according to some embodiments.The sidewalls of the sample well may be tapered or curved. Forming asample well in this manner can admit more excitation energy to theexcitation region, yet still appreciably attenuate excitation energythat travels toward the specimen. Additionally, emission radiated by asample may preferentially radiate toward the end of the sample well withthe larger aperture, because of favorable energy transfer in thatdirection.

In some embodiments, a divot 3-216 may have a smaller transversedimension than the base of the sample well, as depicted in FIG. 3-7B. Asmaller divot may be formed by coating sidewalls of the sample well witha sacrificial layer before etching the divot, and subsequently removingthe sacrificial layer. A smaller divot may be formed to retain a samplein a region that is more equidistant from the conductive walls of thesample well. Retaining a sample equidistant from the walls of the samplewell may reduce undesirable effects of the sample well walls on theradiating sample, e.g., quenching of emission and/or altering ofradiation lifetimes.

FIGS. 3-7C and 3-7D depict another embodiment of a sample well.According to this embodiment, a sample well 2-211 may compriseexcitation-energy-enhancing structures 3-711 and an adherent 3-211formed adjacent the excitation-energy-enhancing structures. Theenergy-enhancing structures 3-711 may comprise surface plasmon ornano-antenna structures formed in conductive materials on an opticallytransparent layer 3-235, according to some embodiments. FIG. 3-7Cdepicts an elevation view of the sample well 2-211 and nearby structure,and FIG. 3-7D depicts a plan view. The excitation-energy-enhancingstructures 3-711 may be shaped and arranged to enhance excitation energyin a small localized region. For example, the structures may includepointed conductors having acute angles at the sample well that increasethe intensity of the excitation energy within an excitation region3-215. In the depicted example, the excitation-energy-enhancingstructures 3-711 are in the form of a bow-tie. Samples 3-101 diffusinginto the region may be retained, temporarily or permanently, by theadherent 3-211 and excited by excitation energy that may be deliveredfrom an excitation source 2-250 located in the instrument 2-120.According to some embodiments, the excitation energy may drivesurface-plasmon currents in the energy-enhancing structures 3-711. Theresulting surface-plasmon currents may produce high electric fields atthe sharp points of the structures 3-711, and these high fields mayexcite a sample retained in the excitation region 3-215. In someembodiments, a sample well 2-211 depicted in FIG. 3-7C may include adivot 3-216.

Another embodiment of a sample well is depicted in FIG. 3-7E, and showsan excitation-energy-enhancing structure 3-720 formed along interiorwalls of the sample well 2-211. The excitation-energy-enhancingstructure 3-720 may comprise a metal or conductor, and may be formedusing an angled (or shadow), directional deposition where the substrateon which the sample well is formed is rotated during the deposition.During the deposition, the base of the sample well 2-211 is obscured bythe upper walls of the well, so that the deposited material does notaccumulate at the base. The resulting structure 3-720 may form an acuteangle 3-722 at the bottom of the structure, and this acute angle of theconductor can enhance excitation energy within the sample well.

In an embodiment as depicted in FIG. 3-7E, the material 3-232 in whichthe sample well is formed need not be a conductor, and may be anysuitable material such as a dielectric material. According to someimplementations, the sample well 2-211 and excitation-energy-enhancingstructure 3-720 may be formed at a blind hole etched into a dielectriclayer 3-235, and a separate layer 3-232 need not be deposited.

In some implementations, a shadow evaporation may be subsequentlyperformed on the structure shown in FIG. 3-7E to deposit a metallic orconductive energy-enhancing structure, e.g., a trapezoidal structure orpointed cone at the base of the sample well, as depicted by the dashedline. The energy-enhancing structure may enhance the excitation energywithin the well via surface plasmons. After the shadow evaporation, aplanarizing process (e.g., a chemical-mechanical polishing step or aplasma etching process) may be performed to remove or etch back thedeposited material at the top of the sample well, while leaving theenergy-enhancing structure within the well.

In some embodiments, a sample well 2-211 may be formed from more than asingle metal layer. FIG. 3-7F illustrates a sample well formed in amulti-layer structure, where different materials may be used for thedifferent layers. According to some embodiments, a sample well 2-211 maybe formed in a first layer 3-232 (which may be a semiconducting orconducting material), a second layer 3-234 (which may be an insulator ordielectric), and a third layer 2-221 (which may be a conductor orsemiconductor). In some embodiments, a degeneratively-dopedsemiconductor or graphene may be used for a layer of the sample well. Insome implementations, a sample well may be formed in two layers, and inother implementations a sample well may be formed in four or morelayers. In some embodiments, multi-layer materials used for forming asample well may be selected to increase surface-plasmon generation at abase of the sample well or suppress surface-plasmon radiation at a topof the well. In some embodiments, multi-layer materials used for forminga sample well may be selected to suppress excitation energy frompropagating beyond the sample well and multi-layer structure into thebulk specimen.

In some embodiments, multi-layer materials used for forming a samplewell may be selected to increase or suppress interfacial excitons whichmay be generated by excitation energy incident on the sample well. Forexample, multi-excitons, such as biexcitons and triexitons, may begenerated at an interface between two different semiconductor layersadjacent a sample well. The sample well may be formed in both the metallayer and the first semiconductor layer such that the interface betweenthe first semiconductor layer and a second semiconductor layer is at anexcitation region 3-215 of the sample well. Interfacial excitons mayhave longer lifetimes than excitons within the volume of a singlesemiconductor layer, increasing the likelihood that the excitons willexcite a sample or tag via FRET or DET. In some embodiments, at leastone quantum dot at which multi-excitons may be excited may be attachedto a bottom of the sample well (e.g., by a linking molecule). Excitonsexcited at a quantum dot may also have longer lifetimes than excitonswithin the volume of a single semiconductor layer. Interfacial excitonsor excitons generated at a quantum dot may increase the rate of FRET orDET, according to some embodiments.

Various materials may be used to form sample wells described in theforegoing embodiments. According to some embodiments, a sample well2-211 may be formed from at least one layer of material 2-221, which maycomprise any one of or a combination of a conductive material, asemiconductor, and an insulator. In some embodiments, the sample well2-211 comprises a highly conductive metallic layer, e.g., gold, silver,aluminum, copper. In some embodiments, the layer 2-221 may comprise amulti-layer stack that includes any one of or a combination of gold,silver, aluminum, copper, titanium, titanium nitride, and chromium. Insome implementations, other metals may be used additionally oralternatively. According to some embodiments, a sample well may comprisean alloy such as AlCu or AlSi.

In some embodiments, the multiple layers of different metals or alloysmay be used to form a sample well. In some implementations, the materialin which the sample well 2-211 is formed may comprise alternating layersof metals and non-metals, e.g., alternating layers of metal and one ormore dielectrics. In some embodiments, the non-metal may include apolymer, such as polyvinyl phosphonic acid or a polyethylene glycol(PEG)-thiol.

A layer 2-221 in which a sample well is formed may be deposited on oradjacent to at least one optically transparent layer 3-235, according tosome embodiments, so that excitation energy (e.g., in the form ofvisible or near-infrared radiation) and emission energy (e.g., in theform of visible or near-infrared radiation) may travel to and from thesample well 2-211 without significant attenuation. For example,excitation energy from an excitation source 2-250 may pass through theat least one optically transparent layer 3-235 to the excitation region3-215, and emission from the sample may pass through the same layer orlayers to the sensor 2-250.

In some embodiments, at least one surface of the sample well 2-211 maybe coated with one or more layers 3-211, 3-280 of material that affectthe action of a sample within the sample well, as depicted in FIG. 3-8.For example, a thin dielectric layer 3-280 (e.g., alumina, titaniumnitride, or silica) may be deposited as a passivating coating onsidewalls of the sample well. Such a coating may be implemented toreduce sample adhesion of a sample outside the excitation region 3-215,or to reduce interaction between a sample and the material 2-221 inwhich the sample well 2-211 is formed. The thickness of a passivatingcoating within the sample well may be between about 5 nm and about 50nm, according to some embodiments.

In some implementations, a material for a coating layer 3-280 may beselected based upon an affinity of a chemical agent for the material, sothat the layer 3-280 may be treated with a chemical or biologicalsubstance to further inhibit adhesion of a sample species to the layer.For example, a coating layer 3-280 may comprise alumina, which may bepassivated with a polyphosphonate passivation layer, according to someembodiments. Additional or alternative coatings and passivating agentsmay be used in some embodiments.

According to some embodiments, at least a bottom surface of the samplewell 2-211 and/or divot 3-216 may be treated with a chemical orbiological adherent 3-211 (e.g., biotin) to promote retention of asample. The sample may be retained permanently or temporarily, e.g., forat least a period of time between about 0.5 milliseconds and about 50milliseconds. In another embodiment, the adherent may promote temporaryretention of a sample 3-101 for longer periods. Any suitable adherentmay be used in various embodiments, and is not limited to biotin.

According to some embodiments, the layer of material 3-235 adjacent thesample well may be selected based upon an affinity of an adherent forthe material of that layer. In some embodiments, passivation of thesample well's side walls may inhibit coating of an adherent on thesidewalls, so that the adherent 3-211 preferentially deposits at thebase of the sample well. In some embodiments, an adherent coating mayextend up a portion of the sample well's sidewalls. In someimplementations, an adherent may be deposited by an anisotropic physicaldeposition process (e.g., evaporation, sputtering), such that theadherent accumulates at the base of a sample well or divot and does notappreciably form on sidewalls of the sample well.

Various fabrication techniques may be employed to fabricate sample wells2-211 for an assay chip. A few example processes are described below,but the invention is not limited to only these examples.

The sample well 2-211 may be formed by any suitable micro- ornano-fabrication process, which may include, but is not limited to,processing steps associated with photolithography, deep-ultravioletphotolithography, immersion photolithography, near-field optical contactphotolithography, EUV lithography, x-ray lithography, nanoimprintlithography, interferometric lithography, step-and-flash lithography,direct-write electron beam lithography, ion beam lithography, ion beammilling, lift-off processing, reactive-ion etching, selective epitaxy,molecular self-assembly, organic synthesis, etc. According to someembodiments, a sample well 2-211 may be formed using photolithographyand lift-off processing. Example fabrication steps associated withlift-off processing of a sample well are depicted in FIG. 3-9A throughFIG. 3-9E. Although fabrication of only a single sample well orstructure at a pixel is typically depicted in the drawings, it will beunderstood that a large number of sample wells or structures may befabricated on a substrate (e.g., at each pixel) in parallel.

According to some embodiments, a layer 3-235 (e.g., an oxide layer) on asubstrate may be covered with an anti-reflection coating (ARC) layer3-910 and photoresist 3-920, as depicted in FIG. 3-9A. The photoresist3-920 may be exposed and patterned using photolithography anddevelopment of the resist. The photoresist 3-920 may be developed toremove exposed portions or unexposed portions (depending on the resisttype), leaving a pillar 3-922 that has a diameter approximately equal toa desired diameter for the sample well, as depicted in FIG. 3-9B. Theheight of the pillar 3-922 may be greater than a desired depth of thesample well.

The pattern of the pillar 3-922 may be transferred to the ARC layer3-910 via anisotropic, reactive ion etching (RIE), for example as shownin FIG. 3-9C. The region may then be coated with at least one material2-221, e.g., a conductor or metal, that is desired to form the samplewell. A portion of the deposited material, or materials, forms a cap3-232 over the pillar 3-922, as depicted in FIG. 3-9D. The photoresist3-920 and ARC layer 3-910 may then be stripped from the substrate, usinga selective removal process (e.g., using a chemical bath with or withoutagitation which dissolves at least the resist and releases or “liftsoff” the cap). If the ARC layer 3-910 remains, it may be stripped fromthe substrate using a selective etch, leaving the sample well 3-210 asshown in FIG. 3-9E. According to some embodiments, the sidewalls 3-214of the sample well may be sloped due to the nature of the deposition ofthe at least one material 2-221.

As used herein, a “selective etch” means an etching process in which anetchant selectively etches one material that is desired to be removed oretched at a higher rate (e.g., at least twice the rate) than the etchantetches other materials which are not intended to be removed.

Because the resist and ARC layer are typically polymer based, they areconsidered soft materials which may not be suitable for forming samplewells having high aspect ratios (e.g., aspect ratios greater than about2:1 with respect to height-to-width). For sample wells having higheraspect ratios, a hard material may be included in the lift-off process.For example, before depositing the ARC layer and photoresist, a layer ofa hard (e.g., an inorganic material) may be deposited. In someembodiments, a layer of titanium or silicon nitride may be deposited.The layer of hard material should exhibit preferential etching over thematerial, or materials, 2-221 in which the sample well is formed. Afterthe photoresist is patterned, a pattern of the pillar may be transferredinto the ARC layer and the underlying hard material 3-930 yielding astructure as depicted in FIG. 3-9F. The photoresist and ARC layer may bethen stripped, the material(s) 2-221 deposited, and a lift-off stepperformed to form the sample well.

According to some embodiments, a lift-off process may be used to form asample well comprising energy-enhancing structures 3-711, as depicted inFIG. 3-7C and FIG. 3-7D.

An alternative process for forming a sample well is depicted in FIGS.3-10A through 3-10D. In this process, the sample well may be directlyetched into at least one material 3-236. For example, at least onematerial 3-236 in which a sample well is to be formed may be depositedon a substrate 3-325. The layer may be covered by an ARC layer 3-910 anda photoresist 3-920, as illustrated in FIG. 3-10A. The photoresist maybe patterned to form a hole having a diameter approximately equal to adesired diameter of the sample well, as depicted in FIG. 3-10B. Thepattern of the hole may be transferred to the ARC layer and through thelayer 3-230 using an anisotropic, reactive ion etch, as shown in FIG.3-10C for example. The resist and ARC layer may be stripped, yielding asample well as depicted in FIG. 3-10D. According to some embodiments,the sidewalls of a sample well formed by etching into the layer ofmaterial 3-230 may be more vertical than sidewalls resulting from alift-off process.

In some embodiments, the photoresist and ARC layer may be used topattern a hard mask (e.g., a silicon nitride or oxide layer, not shown)over the material 3-236. The patterned hole may then be transferred tothe hard mask, which is then used to transfer the pattern into the layerof material 2-221. A hard mask may allow greater etching depths into thelayer of material 2-221, so as to form sample wells of higher aspectratio.

It will be appreciated that lift-off processes and direct etchingfabrication techniques described above may be used to form a sample wellwhen multiple layers of different materials are used to form a stack ofmaterial in which the sample well is formed. An example stack is shownin FIG. 3-11. According to some embodiments, a stack of material may beused to form a sample well to improve coupling of excitation energy tothe excitation region of a sample well, or to reduce transmission orre-radiation of excitation energy into the bulk specimen. For example,an absorbing layer 3-942 may be deposited over a first layer 3-940. Thefirst layer may comprise a metal or metal alloy, and the absorbing layermay comprise a material that inhibits surface plasmons, e.g., amorphoussilicon, TaN, TiN, or Cr. In some implementations, a surface layer 3-944may also be deposited to passivate the surface surrounding the samplewell (e.g., inhibit adhesion of molecules).

Formation of a sample well including a divot 3-216 may be done in anysuitable manner. In some embodiments, a divot may be formed by etchingfurther into an adjacent layer 3-235, and/or any intervening layer orlayers, adjacent the sample well. For example, after forming a samplewell in a layer of material 2-221, that layer 2-221 may be used as anetch mask for patterning a divot, as depicted in FIG. 3-12. For example,the substrate may be subjected to a selective, anisotropic reactive ionetch so that a divot 3-216 may be etched into adjacent layer 3-235. Forexample, in an embodiment where the material 2-221 is metallic and theadjacent layer 3-235 silicon oxide, a reactive-ion plasma etch having afeed gas comprising CHF3 or CF4 may be used to preferentially removeexposed silicon oxide below the sample well and form the divot 3-216. Asused herein, “silicon oxide” generally refers to SiOx and may includesilicon dioxide, for example.

In some embodiments, conditions within the plasma (e.g., bias to thesubstrate and pressure) during an etch may be controlled to determinethe etch profile of the divot 3-216. For example, at low pressure (e.g.,less than about 100 mTorr) and high DC bias (e.g., greater than about20V), the etching may be highly anisotropic and form substantiallystraight and vertical sidewalls of the divot, as depicted in thedrawing. At higher pressures and lower bias, the etching may be moreisotropic yielding tapered and/or curved sidewalls of the divot. In someimplementations, a wet etch may be used to form the divot, which may besubstantially isotropic and form an approximately spherical divot thatmay extend laterally under the material 2-221, up to or beyond thesidewalls of the sample well.

FIG. 3-13A through FIG. 3-13C depict process steps that may be used toform a divot 3-216 having a smaller transverse dimension than the samplewell 2-211 (for example, a divot like that depicted in FIG. 3-7B). Insome implementations, after forming a sample well, a conformalsacrificial layer 3-960 may be deposited over a region including thesample well. According to some embodiments, the sacrificial layer 3-960may be deposited by a vapor deposition process, e.g., chemical vapordeposition (CVD), plasma-enhanced CVD, or atomic layer deposition (ALD).The sacrificial layer may then be etched back using a first anisotropicetch that is selective to the sacrificial layer 3-960, removes the layerfrom horizontal surfaces, leaves side wall coatings 3-962 on walls ofthe sample well, as depicted in FIG. 3-13B. The etch back may beselective and stop on the material 2-221 and adjacent layer 3-235 insome embodiments, or may be a non-selective, timed etch in someembodiments.

A second anisotropic etch that is selective to the adjacent layer 3-235may be executed to etch a divot 3-216 into the adjacent layer asdepicted in FIG. 3-13C. The sacrificial side wall coatings 3-962 maythen optionally be removed by a selective wet or dry etch. The removalof the sidewall coatings open up the sample well to have a largertransverse dimension than the divot 3-216.

According to some embodiments, the sacrificial layer 3-960 may comprisethe same material as the adjacent layer 3-235. In such embodiments, thesecond etch may remove at least some of the side wall coating 3-962 asthe divot is etched into the adjacent layer 3-235. This etch back of theside wall coating can form tapered sidewalls of the divot in someembodiments.

In some implementations, the sacrificial layer 3-960 may be formed from,or include a layer of, a material that is used to passivate thesidewalls of the sample well (e.g., reduce adhesion of samples at thesidewalls of the sample well). At least some of the layer 3-960 may thenbe left on the walls of the sample well after formation of the divot.

According to some embodiments, the formation of the sidewall coatings3-962 occurs after the formation of the divot. In such an embodiment thelayer 3-960 coats the sidewalls of the divot. Such a process may be usedto passivate the sidewalls of the divot and localize the sample at thecenter of the divot.

Process steps associated with depositing an adherent 3-211 at a base ofa sample well 2-211, and a passivation layer 3-280 are depicted in FIGS.3-14A through 3-14D. According to some embodiments, a sample well mayinclude a first passivation layer 3-280 on walls of the sample well. Thefirst passivation layer may be formed, for example, as described abovein connection with FIG. 3-13B or FIG. 3-8. In some embodiments, a firstpassivation layer 3-280 may be formed by any suitable deposition processand etch back. In some embodiments, a first passivation layer may beformed by oxidizing the material 3-230 in which the sample well isformed. For example, the sample well may be formed of aluminum, whichmay be oxidized to create a coating of alumina on sidewalls of thesample well.

An adherent 3-980 or an adherent precursor (e.g., a material whichpreferentially binds an adherent) may be deposited on the substrateusing an anisotropic physical deposition process, e.g., an evaporativedeposition, as depicted in FIG. 3-14A. The adherent or adherentprecursor 3-980 may form an adherent layer 3-211 at the base of thesample well, as depicted in FIG. 3-14B, and may coat an upper surface ofthe material 2-221 in which the sample well is formed. A subsequentangled, directional deposition depicted in FIG. 3-14C (sometimesreferred to as a shadow deposition or shadow evaporation process) may beused to deposit a second passivation layer 2-280 over an upper surfaceof the material 2-221 without covering the adherent layer 3-211. Duringthe shadow deposition process, the substrate may be rotated around anaxis normal to the substrate while the passivation layer precursors3-990 are deposited, so that the second passivation layer 3-280 depositsmore uniformly around an upper rim of the sample well. A resultingstructure is depicted in FIG. 3-14D, according to some embodiments. Asan alternative to depositing the second passivation layer, a planarizingetch (e.g., a CMP step) may be used to remove adherent from an uppersurface of the material 3-230.

According to some implementations, an adherent layer 3-211 may bedeposited centrally at the base of a tapered sample well, as depicted inFIG. 3-15. For example, an adherent, or adherent precursor, may bedirectionally deposited, as depicted in FIG. 3-14A, in a tapered samplewell, formed as described above. Walls of the sample well may bepassivated by an oxidation process before or after deposition of theadherent layer 3-211. Adherent or precursor remaining on a surface ofthe material 2-221 may be passivated as described in connection withFIG. 3-14D. In some embodiments, an adherent on an upper surface of thematerial 2-221 may be removed by a chemical-mechanical polishing step.By forming an adherent layer, or an adherent layer precursor, centrallyat the base of a sample well, deleterious effects on emission from asample (e.g., suppression or quenching of sample radiation from samplewalls, unfavorable radiation distribution from a sample because it isnot located centrally with respect to energy coupling structures formedaround a sample well, adverse effects on luminescent lifetime for asample) may be reduced.

In some embodiments, lift-off patterning, etching, and depositionprocesses used to form the sample well and divot may be compatible withCMOS processes that are used to form integrated CMOS circuits on asensor chip. Accordingly, sensor may be fabricated using conventionalCMOS facilities and fabrication techniques, though custom or specializedfabrication facilities may be used in some implementations.

Variations of the process steps described above may be used to formalternative embodiments of sample wells. For example, a tapered samplewell such as depicted in FIG. 3-7A or FIG. 3-7B may be formed using anangled deposition process depicted in FIG. 3-14C. For the sample well ofFIG. 3-7B, the angle of deposition may be changed during the depositionprocess. For such embodiments, a sample well having substantiallystraight and vertical sidewalls may first be formed, and then additionalmaterial 2-221 deposited by an angled deposition to taper the sidewallsof the sample well.

B. Coupling Excitation Energy to the Sample Well

As illustrated in FIG. 2-1 and FIG. 2-3, excitation energy 2-251 fromthe excitation source 2-250 is guided to the sample well 2-211 usingcomponents of the instrument 2-120 and components of the assay chip2-110. This section describes the components of the assay chip 2-110that may aid in the coupling of excitation energy 2-251 to the samplewell 2-211.

Coupling of energy from an excitation source to a sample well may beimproved or affected by forming excitation-coupling structures withinand/or adjacent a sample well. Excitation-coupling structures maycomprise micro- or nano-scale structures fabricated around a sample wellin some embodiments, or may comprise structures or particles formed at asample well in some embodiments. Excitation-coupling structures mayaffect radiative excitation of a sample in some implementations, and mayaffect non-radiative excitation of a sample in some implementations. Invarious embodiments, radiative excitation-coupling structures mayincrease an intensity of excitation energy within an excitation regionof a sample well. Non-radiative excitation-coupling structures mayimprove and/or alter non-radiative energy-transfer pathways from anexcitation source (which may be radiative or non-radiative) to a sample.

C. Radiative Excitation-Coupling Structures

There are a number of different types of radiative, excitation-couplingstructures that may be used to affect coupling of excitation energy froman excitation source to an excitation region within a sample well. Someradiative coupling structures may be formed of a conductor (e.g.,include a metal layer), and support surface plasmon oscillations thatlocally affect the excitation energy (e.g., locally alter anelectromagnetic field) near and/or within the sample well. In somecases, surface-plasmon structures may enhance the excitation energywithin an excitation region of the sample well by a factor of two ormore. Some radiative coupling structures may alter the phase and/oramplitude of an excitation field to enhance excitation energy within asample well. Various embodiments of radiative excitation-couplingstructures are described in this section.

FIG. 4-1A depicts just one example of a surface-plasmon structure 4-120that may be used to enhance coupling of excitation energy into a samplewell. The drawing depicts a plan view of a region around asurface-plasmon structure 4-120, and represents results of a numericalsimulation of electric field intensity around the structure. The drawingdepicts a surface-plasmon structure comprising three triangular featureshaving sharp apexes that are located in close proximity to a sample well(not shown). According to some embodiments, a surface-plasmon structuremay comprise a metal or conductor (e.g., a patterned thin film of anyone or combination of the following metals or metal alloys: Al, Au, Ag,Ti, TiN). A thickness of the film may be between approximately 10 nm andapproximately 100 nm in some embodiments, though other thicknesses maybe used in other embodiments. A surface-plasmon structure, in someembodiments, may include sharp features 4-110 located in close proximityto a sample well (e.g., within about 100 nm).

FIG. 4-1B depicts a cross-section, elevation view of the surface-plasmonstructure of FIG. 4-1A, taken at the dashed line. The simulation shows alocalized, high-intensity region 4-505 of the excitation energy adjacentan apex of a triangle of the surface-plasmon structure. For thissimulation, the surface-plasmon structure 4-120 was located on adielectric layer 4-135 (silicon dioxide). The surface-plasmon structuretaps energy from an evanescent field of the waveguide, and enhances theintensity at the sample well.

In some embodiments, enhancement of excitation energy by asurface-plasmon structure may be localized to an extent that a deepsample well 2-211 is not needed. For example, if a high-intensity region4-505 is formed having a diameter of approximately 100 nm with a peakintensity value greater than about 80% of the intensity outside theregion, then a deep sample well may not be needed. Only samples withinthe high-intensity region 4-505 would contribute appreciable emissionfor purposes of detection.

When an incident electromagnetic field interacts with a surface-plasmonstructure, surface-wave currents are generated in the structure. Theshape of the structure can affect the intensity and distribution ofthese surface-plasmons. These localized currents can interact with andsignificantly alter and intensify the incident electromagnetic field inthe immediate vicinity of the surface-plasmon structure, e.g., asdepicted by the high-intensity region 4-505 in FIG. 4-1B. In someembodiments, an emitter (e.g., a fluorescing tag) that emits energy neara surface-plasmon structure can have its emission altered by thestructure, so as to alter a far-field radiation pattern from theemitter.

Another embodiment of a surface-plasmon structure 4-122 is depicted inthe plan view of FIG. 4-1C. The illustrated bow-tie structure comprisestwo triangular metallic structures located adjacent a sample well 2-211.The structures may be patterned below a sample well, for example, and/oradjacent an excitation region of the sample well. There may be a gap4-127 between the sample well and sharp features 4-125 of thesurface-plasmon structure, in some implementations. The gap 4-127 may bebetween approximately 10 nm and approximately 200 nm, according to someembodiments. In some implementations, the gap 4-127 may be betweenapproximately 10 nm and approximately 100 nm. The sharp features 4-125may comprise a point or sharp bend in an edge of the surface-plasmonstructure, as depicted in the drawing. The sharp features may have anysuitable shape. In some embodiments a bend radius of a sharp feature4-125 may be less than approximately five wavelengths associated withthe incident excitation energy. In some embodiments a bend radius of asharp feature 4-125 may be less than approximately two wavelengthsassociated with the incident excitation energy. In some embodiments abend radius of a sharp feature 4-125 may be less than approximately fivewavelengths associated with a surface-plasmon wave that is excited bythe incident excitation energy. In some embodiments a bend radius of asharp feature 4-125 may be less than approximately two wavelengthsassociated with a surface-plasmon wave that is excited by the incidentexcitation energy.

According to some embodiments, surface-plasmon structures 4-122 may bepatterned within a sample well 2-211 as illustrated in the elevationview of FIG. 4-1D. In some embodiments, a surface-plasmon structurewithin a sample well may comprise one or more fingers (e.g., metallicfingers) patterned onto sidewalls of the sample well, as depicted in thedrawing. FIG. 4-1E depicts a plan view of the sample well 2-211 showingthe surface-plasmon structures 4-122 formed on sidewalls within thesample well. In some embodiments, the lower ends of thesesurface-plasmon structures 4-122 form sharp features or bends where theelectromagnetic field will be enhanced. The surface-plasmon structures4-122 may, or may not, extend to a base of the sample well.

In some embodiments, the surface-plasmon structures 4-122 may bearranged to affect the polarization of the excitation energy and/oremitted energy from the sample well. For example, a pattern as depictedin FIG. 4-1E may be used to affect a preferred orientation of linear orelliptical excitation polarization and/or a preferred orientation oflinear or elliptical polarization from an emitter within the samplewell.

Surface-plasmon structures may be patterned in shapes other than thosedepicted in FIG. 4-1A through FIG. 4-1E. For example, surface-plasmonstructures may be patterned as regular or periodic structures, asdepicted in FIG. 4-2A, according to some embodiments. For example, asurface-plasmon structure may be patterned is an array of protrudingfeatures 4-210 on a lower surface of a material 2-221 in which thesample well 2-211 is formed. Periodic surface-plasmon structures may beformed in a regular array, for example, a grating, a grid, a lattice, acircular grating, a spiral grating, an elliptical grating, or any othersuitable structure. There may be a substantially uniform spacing sbetween the protrusions 4-210 of a surface-plasmon structure. In someimplementations, the spacing s may have any value between approximately40 nm and approximately 250 nm. According to some embodiments, theprotrusions may have a height h between approximately 20 nm andapproximately 100 nm. In some implementations, the spacing s may benon-uniform or may be chirped (having a decreasing value at largerradial distances). In some embodiments, the protrusions 4-210 of asurface-plasmon structure may be patterned as a Fresnel zone plate.According to some embodiments, a surface-plasmon structure of 4-210 maybe formed adjacent to a transparent layer and/or dielectric layer 3-235.In some embodiments, the spacing between the protrusions 4-210 may beperiodic, while in other embodiments the protrusions 4-210 may beaperiodic.

In some implementations, a surface-plasmon structure 4-212 may be spacedfrom a material 2-221 in which the sample well is formed as depicted inFIG. 4-2B. For example, there may be an intervening dielectric layer4-247 between the surface-plasmon structure 4-212 and the material4-230. According to some embodiments, a surface plasmons structure 4-212may be located adjacent a divot 3-216 of a sample well, as depicted inFIG. 4-2B. For example, a surface-plasmon structure 4-212 may be locatedadjacent sidewalls of a divot 3-216, as depicted in FIG. 4-2B.

FIG. 4-2C illustrates a surface-plasmon structure 4-214 that is formedas a concentric, circular grating. The structure 4-214 may compriseconcentric conducting rings 4-215, according to some embodiments. Therings may be separated by a regular spacing s and have a height h, asdescribed in connection with FIG. 4-2A. According to some embodiments, asample well 4-210 with an optional divot may be located at a center ofthe rings. The circular grating may be patterned adjacent a base of thesample well.

A periodicity of a surface-plasmon structure may be selected to form aresonant structure according to some embodiments. For example a spacings of a surface-plasmon structure may be selected to be approximatelyone-half wavelength of a surface-plasmon wave that is generated in thestructure by the excitation energy. When formed as a resonant structure,a surface-plasmon structure may accumulate and resonate excitationenergy along the direction of the periodic surface-plasmon structure.Such a resonant behavior can intensify electromagnetic energy within asample well, or adjacent a sample well, as depicted in FIG. 4-2D. Whilethe spacing of the surface plasmon structure may be periodic in someembodiments, in other embodiments the spacing may be aperiodic. Usingaperiodic spacing allows the field enhancement to be specificallydesigned for the wavelengths of excitation energy and wavelengths ofemission energy involved. FIG. 4-2D represents numerically simulatedelectromagnetic field results at the base of the sample well and arounda periodic surface-plasmon structure. The surface-plasmon structure4-216 is located adjacent the material 2-221 in which the sample well isformed, and is adjacent a base of a sample well 2-211. Thesurface-plasmon structure may be in the form of a grating or circulargrating that repeats at regular or irregular spacing intervals inregions away from the sample well and outside the simulated region. Forexample, there may be between three and fifty repeated gratingprotrusions of the surface-plasmon structure 4-216. A region of highintensity 4-240 can be seen at the base of the sample well 2-211. Theintensity within this region has been enhanced by more than a factor of2 over the surrounding region just below the surface-plasmon structure.

FIG. 4-2E depicts, in elevation view, an alternative embodiment of aresonant surface-plasmon structure 4-218. According to some embodiments,a surface-plasmon structure may be formed as periodic or aperiodicgrating or grid patterns, and may be patterned in multiple layers 4-247.A sample well 2-211 may be patterned through the multiple layers 4-247and within the resonant surface-plasmon structure 4-218, according tosome embodiments. In some implementations, a resonant surface-plasmonstructure may comprise discrete conductive elements 4-222 is depicted inthe plan view of FIG. 4-2F. In some implementations, a resonantsurface-plasmon structure may comprise a continuous lattice pattern4-250, as depicted in FIG. 4-2G. A dielectric filler 4-252 may belocated in voids of the conductive material 4-250, and a sample well2-211 may be located with a void.

There are a variety of different surface-plasmon structures that may beused to enhance coupling into a sample well or to affect emission from asample within the sample well. FIG. 4-2H depicts, in plan view, yet analternative embodiment of the surface-plasmon structure. A cross-sectionview of the structure is depicted in FIG. 4-2I taken through line 4-2Iof FIG. 4-2H. According to some implementations, a surface-plasmonstructure may comprise an array of discs 4-260 distributed around asample well 2-211. In some embodiments, each disc 4-260 may beapproximately a distance R from the sample well 2-211. However, asillustrated, the distance from each disc 4-260 to the sample well 2-211may vary. Also, the size of each disc 4-260 may be different. In someimplementations, instead of using conductive discs 4-260, asurface-plasmon structure may comprise a conductive layer through whicha distributed pattern of holes are formed. Such a structure may bereferred to as a “nano-antenna.”

A variety of different processes may be used to pattern surface-plasmonstructures adjacent a sample well. FIG. 4-3A through FIG. 4-5E depictstructures associated with process steps that may be used to formsurface-plasmon structures adjacent to a sample well, according to someembodiments. Referring now to FIG. 4-3A, a process for forming asurface-plasmon structure may comprise forming a resist layer 4-310 onan anti-reflective coating (ARC) 4-320 on a masking layer 4-330. Thelayers may be disposed on a transparent dielectric layer 3-235,according to some implementations. The resist layer 4-310 may comprise aphotoresist or an electron- or ion-beam resist that may belithographically patterned. The masking layer 4-330 may comprise a hardmask formed of an inorganic material (e.g., silicon or silica nitride,or any other suitable material), according to some embodiments.

In some implementations, a photolithographic process may be used topattern the resist 4-310 as depicted in FIG. 4-3B. The selected patternmay comprise a layout of protrusions or holes that will be used to forma desired surface-plasmon structure. After development of the resist4-310, regions of the ARC will be exposed, and the pattern may be etchedinto the ARC layer 4-320 and then into the masking layer 4-330. Theresist and ARC may be stripped from the substrate, and a resultingstructure may appear as shown in FIG. 4-3C. The masking layer 4-330 maythen be used as an etch mask, so that the pattern may be transferredinto the underlying dielectric layer 3-235 via a selective anisotropicetch, as depicted in FIG. 4-3D.

A conductive material 2-221, or a layer of materials comprising aconductor, may then be deposited over the region, as illustrated in FIG.4-3E. Any suitable conductive material may be used for forming a surfaceplasmon structure, whether or not it is deposited as a separate layerfrom the material 2-221. For example, in some cases, a first conductivematerial may be deposited as a base layer of material 2-221 in which asurface-plasmon structure is formed. Examples of materials that may beused for forming a surface-plasmon structure include, but are notlimited to, Au, Al, Ti, TiN, Ag, Cu, and alloys or combination layersthereof.

The material 2-221, or layer of materials, may be deposited by anysuitable deposition process, including but not limited to a physicaldeposition process or a chemical vapor deposition process. In someembodiments, the material 2-221 may have a thickness betweenapproximately 80 nm and approximately 300 nm. In some implementations,the material 2-221 may be planarized (e.g., using a CMP process), thoughplanarization is not necessary. A sample well may be formed in thematerial 2-221 using any suitable process described herein in connectionwith fabricating a sample well.

The inventors have recognized that forming a surface-plasmon structureaccording to the steps shown in FIG. 4-3A through FIG. 4-3E may requireaccurate alignment of the sample well to the surface-plasmon structure.For example, a surface-plasmon structure comprising a concentricgrating, as depicted in FIG. 4-2C, may require accurate alignment of thesample well 2-211 to the center of the surface-plasmon structure 4-214.To avoid fabrication difficulties associated with such accuratealignment, self-alignment processes depicted in FIG. 4-4A through FIG.4-5E may be used.

Referring now to FIG. 4-4A, a process for forming a surface-plasmonstructure and sample well that is self-aligned to the surface-plasmonstructure may comprise forming a masking layer 4-410 on a transparentdielectric layer 3-235. The masking layer may comprise a hard maskformed of an inorganic material, such as silicon or silica nitride,according to some embodiments. A thickness of the masking layer 4-410may be approximately equal to a desired height of a sample well 2-212.For example, the thickness of the masking layer may be betweenapproximately 50 nm and approximately 200 nm, according to someembodiments, though other thicknesses may be used in other embodiments.

The masking layer 4-410 may be patterned to create voids 4-430 havingthe desired pattern of a surface-plasmon structure that will bepatterned in the dielectric layer 3-235. The patterning of the maskinglayer 4-410 may be done with any suitable lithography process (e.g.,photolithography, electron-beam lithography, ion-beam lithography, EUVlithography, x-ray lithography). The resulting structure may appear asshown in FIG. 4-4B. The structure may include a central pillar 4-420,which will be used subsequently to form the self-aligned sample well.

A resist 4-440 (e.g., a photoresist) may then be patterned over thepatterned masking layer 4-410, as depicted in FIG. 4-4C. Alignment forpatterning the resist 4-440 (e.g., mask to substrate alignment) need notbe highly accurate, and only requires the resist 4-440 to cover acentral pillar 4-420 and not cover voids 4-430 that will be used to formthe surface-plasmon structure.

A selective anisotropic etch may then be used to etch the dielectriclayer 3-235 and transfer the pattern of the surface-plasmon structureinto the dielectric, as depicted in FIG. 4-4D according to someembodiments. A selective isotropic etch may then be used to remove theexposed portions of the masking layer 4-410. The isotropic etch may be awet etch, for example, though an isotropic dry etch may be used in someembodiments. Because the resist 4-440 covers the central pillar 4-420,the central pillar will not be etched and remain on the substrate, asdepicted in FIG. 4-4E. The resist 4-440 may then be stripped from thesubstrate exposing the pillar 4-420, as depicted in FIG. 4-4F.

According to some embodiments, a metal conductive material 2-221, or astack of materials including a conductive material, may then bedeposited over the region as illustrated in FIG. 4-4G. The centralpillar 4-420 and a cap of deposited material over the pillar may then beremoved by a selective wet etch of the pillar, lifting off the cap. Theremoval of the central pillar leaves a sample well that is self-alignedto the underlying surface-plasmon structure 4-450.

An alternative process may be used to form a sample well that isself-aligned to a surface-plasmon structure, and is depicted in FIG.4-5A through FIG. 4-5E. According to some embodiments, one or moreconductive layers 4-510, 4-520 may be patterned on a transparentdielectric layer 3-235 using any suitable lithography process, asdepicted in FIG. 4-5A. In some implementations, a first layer 4-510 maycomprise aluminum, and a second layer 4-520 may comprise titaniumnitride, though other material combinations may be used in variousembodiments. A total thickness of the one or more layers may beapproximately equivalent to a desired height of the sample well,according to some embodiments. The patterning may form a sample well2-211, and voids 4-525 adjacent the sample well in the one or more metallayers. The voids may be arranged in the pattern of a desiredsurface-plasmon structure.

In some implementations, the dielectric layer 3-235 may be etched totransfer the pattern of the surface-plasmon structure and sample well2-211 into the dielectric layer to form dielectric voids 4-530, asdepicted in FIG. 4-5B. The etch depth of the dielectric voids 4-530 maybe between approximately 20 nm and approximately 150 nm, according tosome embodiments. A resist 4-440 may be patterned to cover the samplewell, as depicted in FIG. 4-5C. Alignment for patterning the resist neednot be highly accurate, and only need cover the sample well withoutcovering adjacent etched regions of the dielectric layer 3-235 that willbe used to form the surface-plasmon structure.

As illustrated in FIG. 4-5D, a conductive material 4-512, or layers ofmaterials including a conductor, may be deposited over the region usingany suitable deposition process. The material 4-512 may fill the etchedregions of the dielectric layer, and may extend above the one or morelayers 4-510, 4-520. The resist 4-440 and the material covering theresist may then be removed according to a lift-off process. Theresulting structure, shown in FIG. 4-5E, leaves a sample well that isself-aligned to the surrounding surface-plasmon structure. The samplewell includes a divot 3-216.

In some embodiments the process depicted in FIG. 4-5A through FIG. 4-5Emay be used to form a sample well that does not have a divot 3-216. Forexample, the resist 4-440 may be patterned over the sample well 2-211before the dielectric layer 3-235 is etched. The dielectric layer 3-235may then be etched, which will transfer the pattern of thesurface-plasmon structure to the dielectric layer but not form a divot.The process may then proceed as illustrated in FIG. 4-5D and FIG. 4-5Eto create a self-aligned sample well having no divot.

Other structures, in addition to or as an alternative to surface-plasmonstructures, may be patterned in the vicinity of the sample well 2-211 toincrease the excitation energy within the sample well. For example somestructures may alter the phase and/or the amplitude of the incidentexcitation field so as to increase the intensity of the excitationenergy within the sample well. FIG. 4-6A depicts a thin lossy film 4-610that may be used to alter the phase and amplitude of incident excitationenergy and increase the intensity of electromagnetic radiation withinthe sample well.

According to some embodiments, a thin lossy film 4-610 may createconstructive interference of the excitation energy, resulting in fieldenhancement within an excitation region of the sample well. FIG. 4-6Bdepicts a numerical simulation of excitation energy incident upon asample well where a thin lossy film 4-610 has been formed immediatelyadjacent the sample well. For the simulation, the sample well has adiameter of approximately 80 nm and is formed in a metallic layer ofgold approximately 200 nm thick. The sample well comprises an SCN, andsuppresses propagation of excitation energy through the sample well. Thethin lossy film 4-610 is approximately 10 nm thick, is formed fromgermanium, and covers an underlying transparent dielectric comprisingsilicon dioxide. The thin lossy film extends across an entrance apertureof the sample well. The simulation shows that the intensity of theexcitation energy is a highest value at the entrance aperture of thesample well. The intensity of the excitation energy in this brightregion 4-620 is more than twice the value of the intensity to the leftand right of the sample well.

A thin lossy film may be made from any suitable material. For example, athin lossy film may be made from a material where the index ofrefraction n is approximately the same order of magnitude as theextinction coefficient k for the material. In some embodiments, a thinlossy film may be made from a material where the index of refraction nis within about two orders of magnitude difference from the value of theextinction coefficient k of the material. Non-limiting examples of suchmaterials at visible wavelengths are germanium and silicon.

A thin lossy film may be any suitable thickness, which may depend upon acharacteristic wavelength, or wavelengths, associated with theexcitation source, or sources. In some embodiments, a thin lossy filmmay be between approximately 1 nm and approximately 45 nm thick. Inother embodiments, a thin lossy film may be between approximately 15 nmand approximately 45 nm thick. In still other embodiments, a thin lossyfilm may be between approximately 1 nm and approximately 20 nm thick.

Effects of a thin lossy film on reflectance from the material 2-221 inwhich a sample well is formed, excitation energy loss within the thinlossy film, and excitation energy loss within the material 2-221 areshown in the graph of FIG. 4-6C. One curve plotted in the graphrepresents a reflectance curve 4-634, and shows how reflectance from thematerial 2-221 and the thin lossy film 4-610 vary as the thickness ofthe thin lossy film changes from 0 nm to 100 nm. The reflectance reachesa minimum value at about 25 nm, according to the simulated embodiment.The reflectance minimum will occur at different thicknesses depending ona characteristic wavelength of the excitation energy and materials usedfor the thin lossy film and material 2-221. In some implementations athickness of thin lossy film is selected such that the reflectance isapproximately at its minimal value. Curve 4-632 represents the loss inthe film as a function of the thin film thickness, and curve 4-636represents the loss in the metal as a function of the thin filmthickness.

In some embodiments, a thin lossy film 4-610 may be spaced from a samplewell 2-211 and material 2-221, as depicted in FIG. 4-6D. For example, athin dielectric layer 4-620 (e.g., a silicon oxide SiOx) may be formedover a thin lossy film, and a sample well 2-211 may be formed adjacentthe dielectric layer 4-620. A thickness of the dielectric layer 4-620may be between approximately 10 nm and approximately 150 nm according tosome embodiments, though other thicknesses may be used in someembodiments.

Although depicted as a single layer, a thin lossy film may comprisemultiple layers of two or more materials. In some implementations, amultilayer stack comprising alternating layers of a thin lossy film4-610 and a dielectric layer 4-620 may be formed adjacent a sample well2-211, as depicted in FIG. 4-6E. A thickness of a thin lossy film 4-610in a stack of layers may be between approximately 5 nm and approximately100 nm, and a thickness of a dielectric layer 4-620 within the stack maybe between approximately 5 nm and approximately 100 nm, according tosome embodiments. In some implementations, the multilayer stack maycomprise a layer of silicon dioxide (4.2 nm thick), a layer of silicon(14.35 nm thick), and a layer of germanium (6.46 nm thick), though otherthicknesses may be used in other embodiments. In some implementations,the multilayer stack may comprise a layer of silicon dioxide(approximately 4.2 nm thick), a layer of silicon (approximately 14.4 nmthick), and a layer of germanium (approximately 6.5 nm thick), thoughother thicknesses may be used in other embodiments.

A thin lossy film may be fabricated from any suitable material thatexhibits at least some loss to the incident radiation. In someembodiments, a thin lossy film may comprise a semiconductor material,for example silicon and germanium, though other materials may be used.In some implementations, a thin lossy film may comprise inorganicmaterial or a metal. In some embodiments, a thin lossy film may comprisean alloy or compound semiconductor. For example, a thin lossy film maycomprise an alloy including Si (57.4% by weight), Ge (25.8% by weight),and SiO2 (16.8% by weight), though other ratios and compositions may beused in other embodiments.

According to some embodiments, a thin lossy film may be formed on thesubstrate using any suitable blanket deposition process, for example, aphysical deposition process, a chemical vapor deposition process, a spinon process, or a combination thereof. In some embodiments, a thin lossyfilm may be treated after deposition, e.g., baked, annealed and/orsubjected to ion implantation.

Other phase/amplitude altering structures may be used additionally oralternatively to enhance excitation energy within the sample well.According to some implementations and as shown in FIG. 4-7A, areflective stack 4-705 may be spaced from a sample well 2-211. In someembodiments, a reflective stack may comprise a dielectric stack ofmaterials having alternating indices of refraction. For example a firstdielectric layer 4-710 may have a first index of refraction, and asecond dielectric layer 4-720 may have a second index of refractiondifferent than the first index of refraction. The reflective stack 4-705may exhibit a high reflectivity for excitation energy in someembodiments, and exhibit a low reflectivity for radiative emission froman emitter within the sample well. For example, a reflective stack 4-705may exhibit a reflectivity greater than approximately 80% for excitationenergy and a reflectivity lower than approximately 40% for emission froma sample, though other reflectivity values may be used in someembodiments. A dielectric layer 4-730 that transmits the excitationenergy may be located between the reflective stack and the sample well.

According to some implementations, a reflective stack 4-705 depicted inFIG. 4-7A may form a resonator with the material 2-221 in which thesample well 2-211 is formed. For example, the reflective stack may bespaced from the material 2-221 by a distance that is approximately equalto one-half the wavelength of the excitation energy within thedielectric material 4-730, or an integral multiple thereof. By forming aresonator, excitation energy may pass through the reflective stack,resonate, and build up in the space between the material 2-221 and thereflective stack 4-705. This can increase excitation intensity withinthe sample well 2-211. For example, the intensity may increase withinthe resonant structure by more than a factor of 2 in some embodiments,and more than a factor of 5 in some embodiments, and yet more than afactor of 10 in some embodiments.

Additional structures may be added in the vicinity of the sample well,as depicted in FIG. 4-7B and FIG. 4-7C. According to some embodiments, adielectric plug 4-740 having a first index of refraction that is higherthan a second index of refraction of the dielectric layer 4-730 may beformed adjacent the sample well 2-211, as depicted in FIG. 4-7B. Theplug may be in the shape of a cylinder having a diameter approximatelyequal to that of the sample well, though other shapes and sizes may beused. Because of its higher refractive index, the dielectric plug 4-740may condense and guide excitation energy toward the sample well.

A dielectric structure, such as the plug 4-740, may be used with orwithout a reflective stack 4-705, according to some embodiments. Such adielectric structure may be referred to as a dielectric resonantantenna. The dielectric resonant antenna may have any suitable shape,for example, cylindrical, rectangular, square, polygon old, trapezoidal,or pyramid.

FIG. 4-7C and FIG. 4-7D depict a photonic bandgap (PBG) structure thatmay be formed in the vicinity of a sample well 2-211, according to someembodiments. A photonic bandgap structure may comprise a regular arrayor lattice of optical contrast structures 4-750. The optical contraststructures may comprise dielectric material having a refractive indexthat is different from a refractive index of the surrounding dielectricmaterial, according to some embodiments. In some implementations, theoptical contrast structures 4-750 may have a loss value that isdifferent from the surrounding medium. In some implementations, a samplewell 2-211 may be located at a defect in the lattice as depicted in FIG.4-7D. According to various embodiments, the defect in the photoniclattice may confine photons within the region of the defect can enhancethe intensity of the excitation energy at the sample well. Theconfinement due to the photonic bandgap structure may be substantiallyin two dimensions transverse to a surface of the substrate. Whencombined with the reflective stack 4-705, confinement may be in threedimensions at the sample well. In some embodiments, a photonic bandgapstructure may be used without a reflective stack.

Various methods have been contemplated for fabricating theexcitation-coupling structures depicted in FIG. 4-6A through FIG. 4-7D.Structures that require thin planar films (e.g., dielectric films ofalternating refractive index) may be formed by planar depositionprocesses, according to some embodiments. Planar deposition processesmay comprise physical deposition (for example, electron beam evaporationor sputtering) or chemical vapor deposition processes. Structures thatrequire discrete embedded dielectrics formed in three-dimensionalshapes, such as a dielectric resonant antenna 4-740 shown in FIG. 4-7Bor the optical contrast structures 4-750 shown in FIG. 4-7C, may beformed using lithographic patterning and etching processes to etch thepattern into the substrate, and using subsequent deposition of adielectric layer, and a planarization of the substrate, for example.Also contemplated are self-alignment processing techniques for formingdielectric resonant antennas as well as photonic bandgap structures inthe vicinity of the sample well 2-211.

FIG. 4-8A through FIG. 4-8G depict structures associated with processsteps for just one self-alignment process that may be used to form aphotonic bandgap structure and a self-aligned sample well as illustratedin FIG. 4-7C. According to some embodiments, a reflective stack 4-705may be first formed on a substrate above a dielectric layer 3-235, asillustrated in FIG. 4-8A. A second dielectric layer 4-730 may then bedeposited over the reflective stack. The thickness of the dielectriclayer 4-730 may be approximately equal to about one-half a wavelength ofthe excitation energy in the material, or an integral multiple thereof.Process steps described in connection with FIG. 4-4A through FIG. 4-4Emay then be carried out to form a pillar 4-420 above the dielectriclayer 4-730 and a pattern of etched features 4-810 for the photonicbandgap structure. The etched features may extend into the dielectriclayer 4-730 and optionally into the reflective stack 4-705. Theresulting structure may appear as shown in FIG. 4-8A.

A resist 4-440 covering the pillar 4-420 may be stripped from thesubstrate and a conformal deposition performed to fill the etchedfeatures with a filling material 4-820, as depicted in FIG. 4-8B. Thefilling material 4-820 may be the same material that is used to form thepillar 4-420, according to some embodiments. For example the fillingmaterial 4-820 and the pillar 4-420 may be formed of silicon nitride andthe dielectric layer 4-730 may comprise an oxide, e.g., SiO₂.

An anisotropic etch may then be carried out to etch back the fillingmaterial 4-820. The filling material may be etched back to expose asurface of the dielectric layer 4-730, according to some embodiments,resulting in a structure as depicted in FIG. 4-8C. The etch may leave apillar 4-830 comprising the original pillar 4-420 and sidewalls 4-822that remain from the filling material 4-820.

A resist 4-440 may then be patterned over the substrate as depicted inFIG. 4-8D. For example, the resist may be coated onto the substrate, ahole patterned in the resist, and the resist developed to open up aregion in the resist around the pillar 4-830. Alignment of the hole tothe pillar need not be highly accurate, and only need expose the pillar4-830 without exposing the underlying photonic bandgap structuresembedded in the dielectric layer 4-730.

After the pillar 4-830 is exposed, and isotropic etch may be used toreduce the transverse dimension of the pillar. According to someembodiments, the resulting pillar shape may appear as depicted in FIG.4-8E. The resist 4-440 may then be stripped from the substrate and amaterial 2-221, or layers of materials, may be deposited over theregion. In some embodiments, the material 2-221 may be etched back usinga CMP process to planarize the region as depicted in FIG. 4-8F.Subsequently, a selective dry or wet etch may be used to remove theremaining pillar structure leaving a sample well 2-211, as illustratedin FIG. 4-8G. As indicated by the drawings, the sample well 2-211 isself-aligned to the photonic bandgap structure patterned in thedielectric layer 4-730.

As an alternative process, the filling material 4-820 may comprise adifferent material than the material used to form the pillar 4-420. Inthis process, the steps associated with FIG. 4-8D and FIG. 4-8E may beomitted. After deposition of material 2-221 and planarization, asdepicted in FIG. 4-8F, a selective etch may be performed to remove thepillar 4-420. This may leave sidewalls of the filling material 4-820lining the sample well 2-211.

D. Non-Radiative Excitation-Coupling Structures

The present disclosure provides structures for non-radiative coupling ofexcitation energy to a sample within the sample well. Just oneembodiment of a non-radiative coupling structure is depicted in FIG.4-9A. According to some embodiments, a non-radiative coupling structuremay comprise a semiconductor layer 4-910 formed immediately adjacent asample well 2-211. The semiconductor layer 4-910 may be an organicsemiconductor in some embodiments, or an inorganic semiconductor in someembodiments. In some implementations, a divot 3-216 may, or may not, beformed in the semiconductor layer. The semiconductor layer 4-910 mayhave a thickness between approximately 5 nm and approximately 100 nmaccording to some embodiments, though other thicknesses may be used insome embodiments. According to some implementations, excitation energyor photons 4-930 from an excitation source may impinge upon thesemiconductor layer 4-910 and produce excitons 4-920. The excitons maydiffuse to a surface of the sample well where they may non-radiativelyrecombine and transfer energy to a sample adjacent the walls of thesample well.

FIG. 4-9B depicts another embodiment in which a semiconductor layer4-912 may be used to non-radiatively transfer energy from excitationenergy to a sample. In some embodiments, a semiconductor layer 4-912 maybe formed at the bottom of a sample well or in a divot of the samplewell 2-211, as depicted in the drawing. The semiconductor layer 4-912may be formed in a sample well by using a directional deposition processas described herein in connection with process steps for depositing anadherent at the base of the sample well, according to some embodiments.The semiconductor layer 4-912 may have a thickness between approximately5 nm and approximately 100 nm according to some embodiments, thoughother thicknesses may be used in other embodiments. Incident radiationmay generate excitons within the semiconductor layer, which may thendiffuse to the a bottom surface of the sample well 2-211. The excitonsmay then non-radiatively transfer energy to a sample within the samplewell.

The present disclosure also provides multiple non-radiative pathways fortransferring excitation energy to a sample. According to someembodiments, and as depicted in FIG. 4-9C, an energy-transfer particle4-940 may be deposited within a sample well. The energy-transferparticle may comprise a quantum dot in some embodiments, or may comprisea molecule in some embodiments. In some implementations, theenergy-transfer particle 4-940 may be functionalized to a surface of thesample well through a linking molecule. A thin semiconductor layer 4-910may be formed adjacent the sample well, or within the sample well, andexcitons may be generated within the semiconductor layer from theexcitation energy incident upon the semiconductor layer, as depicted inthe drawing. The excitons may diffuse to the surface of the sample well,and non-radiatively transfer energy to the energy-transfer particle4-940. The energy-transfer particle 4-940 may then non-radiativelytransfer energy to a sample 3-101 within the sample well.

According to some implementations, there may be more than oneenergy-transfer particle 4-940 within a sample well. For example, alayer of energy-transfer particles 4-942 may be deposited within asample well, such as the sample well depicted in FIG. 4-9C.

In some implementations, energy-transfer particles 4-942, or a singleenergy-transfer particle 4-940, may be deposited at a base of a samplewell, as depicted in FIG. 4-9D. The energy-transfer particle, orparticles, may radiatively or non-radiatively transfer excitation energyto a sample 3-101 within the well. For example, an energy-transferparticle may absorb incident energy to form an excited state of theenergy-transfer particle, and then radiatively or non-radiativelytransfer energy to the sample 3-101.

In some implementations, an energy-transfer particle may absorb incidentexcitation energy, and then re-emit radiative energy at a wavelengththat is different than the wavelength of the absorbed excitation energy.The re-emitted energy may then be used to excite a sample within thesample well. FIG. 4-9E represents spectral graphs associated with adown-converting energy-transfer particle. According to some embodiments,a down-converting energy-transfer particle comprises a quantum dot thatmay absorb short wavelength radiation (higher energy), and emit one ormore longer wavelength radiations (lower energy). An example absorptioncurve 4-952 is depicted in the graph as a dashed line for a quantum dothaving a radius between 6 to 7 nm. The quantum dot may emit a first bandof radiation illustrated by the curve 4-954, a second band of radiationillustrated by the curve 4-956, and a third band of radiationillustrated by the curve 4-958.

In some implementations an energy-transfer particle may up convertenergy from an excitation source. FIG. 4-9F depicts spectra associatedwith up conversion from an energy-transfer particle. According to someembodiments, a quantum dot may be excited with radiation atapproximately 980 nm, and then re-emit into one of three spectral bandsas illustrated in the graph. A first band may be centered atapproximately 483 nm, a second band may be centered at approximately 538nm, and a third band may be centered at approximately 642 nm. There-emitted photons from the quantum dot are more energetic than thephotons of the radiation used to excite the quantum dot. Accordingly,energy from the excitation source is up-converted. One or more of theemitted spectral bands may be used to excite one or more one or moresamples within the sample well.

E. Directing Emission Energy Towards the Sensor

The assay chip 2-110 may include one or more components per pixel toimprove collection of emission energy by the sensors on the instrument.Such components may be designed to spatially direct emission energytowards the sensors and increase the directionality of the emissionenergy from the sample well 2-211. Both surface optics and far-fieldoptics may be used to direct the emission energy towards the sensor.

1. Surface Optics

Components within a pixel of the assay chip 2-110 located near thesample well of the pixel may be configured to couple with the emissionenergy emitted by a sample. Such components may be formed at theinterface between two layers of the assay chip. For example, someemission energy coupling elements may be formed at the interface betweena sample well layer and the layer adjacent to the sample well layeropposite to where the sample wells are formed. In some instances, thelayer underneath the sample well layer is a dielectric layer and theemission energy coupling elements may support surface plasmons. In otherembodiments, the sample well layer may be a conductive material adjacentto an optically-transparent material. Surface-energy coupling elementsmay be surface optical structures that are excited by and interact withradiative emission from the sample well.

A characteristic dimension of a surface optical structure such as agrating period, feature size, or distance from the sample well may beselected to maximally couple a parallel component of an emission energymomentum vector into a surface wave momentum vector for a surfaceplasmon. For example, the parallel component of the emission energymomentum vector may be matched to the surface wave momentum vector for asurface plasmon supported by the structure, according to someembodiments. In some embodiments, a distance d from the sample well toan edge or characteristic feature of a surface optical structure may beselected so as to direct emission energy from the sample well in aselected direction, such as normal to the surface or inclined at anangle θ from normal to the surface. For example, the distance, d, may bean integral number of surface-plasmon wavelengths for directing emissionnormal to the surface. In some embodiments, distance, d, may be selectedto be a fractional surface-plasmon wavelength, or wavelength modulothereof.

According to some embodiments, the surface optical structures may directradiative emission energy from a sample well in a direction normal tothe sample well layer. The coupled energy may be directed in the normaldirection in a narrowed, directional radiation pattern.

An example of a surface optical structure is a concentric grating. Aconcentric grating structure that may be formed in a pixel of the assaychip to direct emission energy towards one or more sensors of the pixel.The concentric grating structure may be formed around a sample well. Anexample of a concentric circular grating surface 5-102 as a surfaceplasmon structure is depicted in FIG. 5-1. The circular grating maycomprise any suitable number of rings and the number of rings (six)shown in FIG. 10-1 is a non-limiting example. The circular grating maycomprise protruding rings from a surface of a conductive layer. Forexample, the circular grating may be formed at the interface of thesample well layer and a dielectric layer formed underneath the samplewell layer. The sample well layer may be a conductive material and theconcentric grating may be formed by patterning the grating structure atthe interface between the conductive material and the dielectric. Therings of the circular grating may be on a regular periodic spacing, ormay have irregular or aperiodic spacings between the rings. The samplewell may be located at or near the center of the circular grating. Insome embodiments, the sample well may be located off-center to thecircular grating and may be positioned a certain distance from thecenter of the grating. In some embodiments, a grating-type surfaceenergy-coupling component may comprise a spiral grating. An example of aspiral grating 5-202 is depicted in FIG. 5-2. The spiral grating 5-202may comprise a spiral aperture in a conductive film. Any suitabledimensions of the spiral grating may be used to form the spiral grating.

A concentric grating may have any suitable number of rings and be of anysuitable size By way of example and not limitation, a concentric gratingmay have six rings with radii having approximately 234 nm, approximately606 nm, approximately 1005 nm, approximately 1397 nm, approximately 1791nm, and approximately 2186 nm. In other embodiments, the concentric ringmay have two, three, four or eight rings.

FIG. 5-3A illustrates a radiation pattern 5-302 for emission energy fromthe sample well 2-211. The concentric grating structure 2-223 causes theemission energy to have greater directionality compared to the radiationpattern formed in the absence of the grating structure 2-223. In someembodiments, the emission energy is directed downward, normal to themetal layer 2-221. The radiation pattern may focus the emission energysuch that the majority of the light is focused in substantially the samedirection toward the sensor. In some embodiments, the radiation patternmay focus the luminescence such that the majority of the luminescenceforms a narrow column-like shape centered below the target volume anddirected downward towards the sensors. In some embodiments, a concentricgrating may provide a directivity of more than 10 for 1 ring, more than15 for 2 rings, more than 18 for 3 rings, more than 20 for four rings,or more than 15 for 1 ring, as shown by the plot in FIG. 5-3B

Another example of a surface optic or surface plasmon structure is anano-antenna structure. A nano-antenna structure may be designed tospatially direct emission energy from the sample well. In someembodiments, the location of the sample well with respect to thenano-antenna structure is selected so as to direct the emission energyfrom the sample well in a particular direction towards one or moresensors. Nano-antennas may comprise nano-scale dipole antenna structuresthat are designed to produce a directional radiation pattern whenexcited by emission energy. The nano-antennas may be distributed arounda sample well. The directional radiation pattern may result from asummation of the antennas' electromagnetic fields. In some embodiments,the directional radiation pattern may result from a summation of theantennas' electromagnetic fields with the field emitted directly fromthe sample. In some implementations, the field emitted directly from thesample may be mediated by a surface plasmon between the sample well andnano-antenna structure.

The dimensions of the individual nano-antennas that form thenano-antenna structure may be selected for the combined ability of theoverall nano-antenna structure to produce specific distributionpatterns. For example, the diameters of the individual nano-antennas mayvary within a nano-antenna structure. However, in some instances, thediameters may be the same within a set of nano-antennas. In otherimplementations, a few selected diameters may be used throughout theoverall nano-antenna structure. Some nano-antennas may be distributed ona circle of radius R and some may be shifted in a radial direction fromthe circle. Some nano-antennas may be equally spaced around a circle ofradius R (e.g., centered on equivalent polar-angle increments), and somemay be shifted from equal spacing around the circle. In someembodiments, the nano-antennas may be arranged in a spiral configurationaround a sample well. Additionally or alternatively, otherconfigurations of nano-antennas are possible, such as a matrix arrayaround the sample well, a cross distribution, and star distributions.Individual nano-antennas may be shapes other than a circle, such assquare, rectangular, cross, triangle, bow-tie, annular ring, pentagon,hexagon, polygons, etc. In some embodiments, the circumference of anaperture or disc may be approximately an integer multiple of afractional wavelength, e.g., (N/2)λ.

A nano-antenna array may direct emission energy from a sample intoconcentrated radiation lobes. When a sample emits energy, it may excitesurface plasmons that propagate from the sample well to thenano-antennas distributed around the sample well. The surface plasmonsmay then excite radiation modes or dipole emitters at the nano-antennasthat emit radiation perpendicular to the surface of the sample welllayer. The phase of an excited mode or dipole at a nano-antenna willdepend upon the distance of the nano-antenna from the sample well.Selecting the distance between the sample well and an individualnano-antenna controls the phase of radiation emitted from thenano-antenna. The spatial radiation mode excited at a nano-antenna willdepend upon the geometry and/or size of the nano-antenna. Selecting thesize and/or geometry of an individual nano-antenna controls the spatialradiation mode emitted from the nano-antenna. Contributions from allnano-antennas in the array and, in some instances the sample well, maydetermine an overall radiation lobe or lobes that form the radiationpattern. As may be appreciated, phase and spatial radiation mode emittedfrom an individual nano-antenna may depend upon wavelength, so that theoverall radiation lobe or lobes that form the radiation pattern willalso be dependent upon wavelength. Numerical simulations of theelectromagnetic fields may be employed to determine overall radiationlobe patterns for emission energies of different characteristicwavelengths.

The nano-antenna may comprise an array of holes or apertures in aconductive film. For example, the nano-antenna structure may be formedat the interface between a conductive sample well layer and anunderlying dielectric layer. The holes may comprise sets of holesdistributed in concentric circles surrounding a central point. In someembodiments, a sample well is located at the central point of the array,while in other embodiments the sample well may be off-center. Eachcircularly-distributed set of holes may comprise a collection ofdifferent diameters arranged smallest to largest around the circulardistribution. The hole diameters may be different between the sets(e.g., a smallest hole in one set may be larger than a smallest hole inanother set), and the location of the smallest hole may be oriented at adifferent polar angle for each set of circles. In some embodiments,there may be one to seven sets of the circularly-distributed holes in anano-antenna. In other embodiments, there may be more than seven sets.In some embodiments, the holes may not be circular, but may be anysuitable shape. For example, the holes may be ellipses, triangles,rectangles, etc. In other embodiments, the distribution of holes may notbe circular, but may create a spiral shape.

FIGS. 5-4A and 5-4B illustrate an exemplary nano-antenna structurecomprised of holes or apertures in a conductive layer. FIG. 5-4A shows atop planar view of the surface of an assay chip with a sample well 5-108surrounded by holes 5-122. The nano-antenna holes are distributed withtheir centers approximately around a circle of radius R. In thisnon-limiting example, the hole diameters vary by incrementallyincreasing around the circumference of the circle of holes. FIG. 5-4Bshows a schematic of a cross-sectional view of the assay chip shown inFIG. 5-4A along line B-B′. The sample well layer 5-116 that includessample well 5-108 and apertures 5-122 that are part of the nano-antennastructure. Layer 5-118 of the assay chip lies underneath sample welllayer 5-116. Layer 5-118 may be a dielectric material and/or anoptically transparent material.

In some embodiments, the nano-antenna structure may comprise a pluralityof disks. The disks of the nano-antenna structure may be formed asconductive disks protruding from a surface of a conductive material. Theconductive material may be adjacent an optically-transparent material.In some embodiments, the nano-antennas may be distributed around asample well. In some instances, the nano-antennas may be distributedapproximately around a sample well at a circle of radius R. Anano-antenna array may comprise multiple sets of nano-antennasdistributed approximately on additional circles of different radiiaround a sample well.

FIGS. 5-5A and 5-5B illustrate an exemplary embodiment of a nano-antennastructure comprising disks protruding from a conductive layer. FIG. 5-5Ashows a top planar view schematic of the surface of an assay chip with asample well 5-208 surrounded by disks 5-224. The nano-antenna disks aredistributed approximately around a circle of radius R. In thisnon-limiting example, two diameters are used for the disks and the disksalternate between these two diameters around the circumference of thecircle of nano-antenna. FIG. 5-5B shows a schematic of a cross-sectionalview of the assay chip shown in FIG. 5-5A along line C-C′. The samplewell layer 5-216 that includes sample well 5-208 and disks 5-224 thatare part of the nano-antenna structure. The disks 5-224 protrude fromthe sample well layer 5-216 by a certain distance. In some embodiments,the distance the disks extend from the sample well layer may vary withina nano-antenna structure. Layer 5-218 of the assay chip lies underneathsample well layer 5-216. Layer 5-18 may be a dielectric material and/oran optically transparent material. The sample well layer 5-216 and theprotruding disks may be a conductive material.

In some embodiments a nano-antenna structure may be centered proximateto a sample well. The nano-antenna may take any suitable shape. By wayof example and not limitation, the nano-antenna may be a symmetricalshape such as a cylinder, disk or cube. The nano-antenna may be made ofany suitable dielectric material, for example, silicon nitride.Alternatively, titanium oxide may be used. The nano-antenna dimensionsmay be depend on the dielectric material used and may be tailored toperform the desired enhancement. In some embodiments, the nano-antennamay have a width of approximately 800 nm and a depth of approximately1050 nm. The nano-antenna may the radiation pattern of emission energyfrom a sample well such that the majority of the light is focused insubstantially the same direction, such as in a narrow column-like shapecentered below the target volume and the corresponding nano-antenna anddirected downward towards the sensors, as illustrated in FIG. 5-5C. Insome embodiments, a directivity of 39.7 is predicted.

In addition to focusing the luminescence toward the sensor, thenano-antenna may enhance the excitation energy into the sample well.Excitation energy from below the nano-antenna and directed toward thetarget volume enters the nano-antenna and is largely concentrated at thetarget volume. In some embodiments, an enhancement of 57.4 is predictedat the sample well for a nano-antenna of a width of 400 nm and a heightof 1050 μm

2. Far Field Optics

In some embodiments, the layer directly under the surface optics may bea spacer layer 2-225 of any suitable thickness and be made of anysuitable dielectric material. The spacer layer may be, for example, 10μm in thickness and may be made of silicon dioxide. Alternatively, thisspacer layer may be 48 μm or 50 μm. The under the spacer layer may beone or more lens layers with additional spacer layers. For example, FIG.5-6A illustrates an upper lens layer 5-601 which may include at leastone refractive lens. In some embodiments, the upper lens layer may belocated 5 m below the sample well layer 2-221. There may be one or morelenses associated with each sample well. In some embodiments, a lensarray may be used, such as a refractive lens array. In some embodiments,each lens of the upper lens layer 5-601 is centered below sample well2-211 and may have a radius, for example, smaller than 10.5 μm. Theupper lens layer may be made of any suitable dielectric material suchas, by way of example and not limitation, silicon nitride.

In some embodiments, the dimensions of the upper lens layer are asfollows: d1 may be approximately 12 m; d2 may be approximately 2 m;distance between d1 on neighboring lenses may be approximately 20.966 m;and the radius of curvature of the lens may be approximately 20.35 m.Alternatively, d1 may be approximately 8 m and d2 may be approximately 6m.

The layer directly under the upper lens layer may be a structural and/oroptical layer 5-605 made of any suitable dielectric. This structuraland/or optical layer 5-605 may be made of silicon dioxide in the form offused silica. The layer directly under the structural layer may be alower lens layer 5-603 which may include at least one additional lens.In some embodiments, each lens in the lower lens layer 5-603 may also becentered below the sample well. The lower lens layer 5-603 may be madeof any suitable dielectric material such as, by way of example and notlimitation, silicon nitride. The distance from the top of the upper lenslayer to the bottom of the lower lens layer may be 100-500 μm. The layerdirectly under the lower lens layer may include an anti-reflection layerthat passes both excitation energy and the emission energy and reducesthe amount of light reflected. The layer directly under theanti-reflection layer may include structural components to allow thechip to align with and mount onto the instrument. The layer directlyunder the chip-mounting layer may include a protective cover to protectthe system from damage and contamination, including dust.

While FIG. 5-6A illustrates two lens layers using refractive lenses, anysuitable lens may be used. For example, Fresnel lenses, microlenses,refractive lens pairs and/or flat lenses may be used. FIG. 5-6Billustrates an embodiment using Fresnel lenses in both an upper lenslayer 5-611 and a lower lens layer 5-613, separated by a structuraland/or optical layer 5-605.

In some embodiments, any of the interfaces between the layers describedabove in the chip may include an anti-reflection coating oranti-reflection layer. Both the upper lens layer and the second lenslayer may be arranged below the sample well to focus the luminescenceemitted from the array of sample wells into a relay lens of theinstrument. The distance from the bottom of the sample well layer to afocal point of the luminescence from the target volumes may beapproximately 30.3 mm, for example. This focal point may occur withinthe relay lens, for example, at a longitudinal center point of the relaylens.

III. INSTRUMENT COMPONENTS

A. Microscopy Layer of the Instrument

In some embodiments, the instrument may include a microscopy layer whichmay include sub-layers as illustrated in FIG. 6-1. In particular, themicroscopy layer may include a sub-layer that includes a polychroicmirror 2-230 tilted at an angle θ to direct the excitation energy towardthe assay chip. This polychroic mirror may be substantially dielectric,and reflects the excitation energy while substantially transmitting theemission energy from the sample in one or more of the sample wells onthe assay chip. Optionally, an astigmatism compensation element 6-101that includes an additional dielectric layer may be provided underneaththe polychroic mirror and tilted at the same angle θ, but about an axisthat is orthogonal to that of the polychroic mirror's tilt, to providecompensation for astigmatism introduced by the polychroic mirror. InFIG. 6-1, the astigmatism compensation element 6-101 is illustrated astilted in the same plane as the top filter, but it should be appreciatedthat the illustration represents a tilting with respect to the topfilter and it is not meant to limit the orientation of the astigmatismcompensation element 6-101 in any way. This astigmatism compensationelement 6-101 may also provide additional filtering. For example, theastigmatism compensation element 6-101 may be another polychroic mirrorthat further filters the excitation energy while transmitting theemission energy. A lens 6-103 may be provided underneath the astigmatismcompensation element 6-101 to further help process the emission energyfrom the sample wells. The lens 6-103 may be, for example, 25.4 m indiameter, but any suitable diameter may be used. In some embodiments,the lens is a relay lens comprising a plurality of lens elements. Forexample, the relay lens may include six separate lens elements. In someembodiments, the relay lens may be, approximately 17.5 mm in length.Additional filtering elements may be used before or after lens 6-103 tofurther reject the excitation energy to prevent it from reaching thesensors.

B. Sensor Chip

Emission energy emitted from a sample in the sample well may betransmitted to the sensor of a pixel in a variety of ways, some examplesof which are described in detail below. Some embodiments may use opticaland/or plasmonic components to increase the likelihood that light of aparticular wavelength is directed to an area or portion of the sensorthat is dedicated to detecting light of that particular wavelength. Thesensor may include multiple sub-sensors for simultaneously detectingemission energy of different wavelengths.

FIG. 6-2A is a schematic diagram of a single pixel of the sensor chipaccording to some embodiments where at least one sorting element 6-127is used to direct emission energy of a particular wavelength to arespective sub-sensor 6-111, 6-112, 6-113, and 6-114. The emissionenergy 2-253 travels from a sample well through the assay chip and theoptical system of the instrument until it reaches a sorting element6-127 of the sensor chip. The sorting element 6-127 couples thewavelength of the emission energy 2-253 to a spatial degree of freedom,thereby separating the emission energy into its constituent wavelengthcomponents, referred to as sorted emission energy. FIG. 6-2A illustratesschematically the emission energy 2-253 being split into four sortedemission energy paths through a dielectric material 6-129, each of thefour paths associated with a sub-sensor 6-111 through 6-114 of thepixel. In this way, each sub-sensor is associated with a differentportion of the spectrum, forming a spectrometer for each pixel of thesensor chip.

Any suitable sorting element 6-127 may be used to separate the differentwavelengths of the emission energy. Embodiments may use optical orplasmonic elements. Examples of optical sorting elements include, butare not limited to, holographic gratings, phase mask gratings, amplitudemask gratings, and offset Fresnel lenses. Examples of plasmonic sortingelements include, but are not limited to phased nano-antenna arrays, andplasmonic quasi-crystals.

FIG. 6-2B is a schematic diagram of a single pixel of the sensor chipaccording to some embodiments where filtering elements 6-121, 6-122,6-123 and 6-124 are used to direct emission energy of a particularwavelength to a respective sub-sensor and prevent emission energy ofother wavelengths from reaching the other sub-sensors. The emissionenergy 2-253 travels from a sample well through the assay chip and theoptical system of the instrument until it reaches one of the filteringelements 6-121 through 6-124. The filtering elements 6-121 through6-124, each associated with a particular sub-sensor 6-111 through 6-114,are each configured to transmit emission energy of a respectivewavelength and reject emission energy of other wavelengths by absorbingthe emission energy (not illustrated in FIG. 6-1B) and/or reflecting theemission energy. After passing through a respective filtering element,the filtered emission energy travels through a dielectric material 6-129and impinges on a corresponding sub-sensor 6-111 through 6-114 of thepixel. In this way, each sub-sensor is associated with a differentportion of the spectrum, forming a spectrometer for each pixel of thesensor chip.

Any suitable filtering elements may be used to separate the differentwavelengths of the emission energy. Embodiments may use optical orplasmonic filtering elements. Examples of optical sorting elementsinclude, but are not limited to, reflective multilayer dielectricfilters or absorptive filters. Examples of plasmonic sorting elementsinclude, but are not limited to frequency selective surfaces designed totransmit energy at a particular wavelength and photonic band-gapcrystals.

Alternatively, or in addition to the above mentioned sorting elementsand filtering elements, additional filtering elements may be placeadjacent to each sub-sensor 6-111 through 6-114. The additionalfiltering elements may include a thin lossy film configured to createconstructive interference for emission energy of a particularwavelength. The thin lossy film may be a single or multi-layer film. Thethin lossy film may be made from any suitable material. For example, thethin lossy film may be made from a material where the index ofrefraction n is approximately the same order of magnitude as theextinction coefficient k. In other embodiments, the thin lossy film maybe made from a material where the index of refraction n is within abouttwo orders of magnitude difference from the value of the extinctioncoefficient k of the material. Non-limiting examples of such materialsat visible wavelengths are germanium and silicon.

The thin lossy film may be any suitable thickness. In some embodiments,the thin lossy film may be 1-45 nm thick. In other embodiments, the thinlossy film may be 15-45 nm thick. In still other embodiments, the thinlossy film may be 1-20 nm thick. FIG. 6-3A illustrates an embodimentwhere the thin lossy films 6-211 through 6-214 each have a differentthickness determined at least in part by the wavelength that isassociated with each sub-sensor 6-11 through 6-114. The thickness of thefilm determines, at least in part, a distinct wavelength that willselectively pass through the thin lossy film to the sub-sensor. Asillustrated in FIG. 6-211, thin lossy film 6-211 has a thickness d1,thin lossy film 6-212 has a thickness d2, thin lossy film 6-213 has athickness d3, and thin lossy film 6-214 has a thickness d4. Thethickness of each subsequent thin lossy film is less than the previousthin lossy film such that d1>d2>d3>d4.

Additionally, or alternatively, the thin lossy films may be formed of adifferent material with a different properties such that emission energyof different wavelengths constructively interfere at each respectivesub-sensor. For example, the index of refraction n and/or the extinctioncoefficient k may be selected to optimize transmission of emissionenergy of a particular wavelength. FIG. 6-3B illustrates thin lossyfilms 6-221, 6-222, 6-223 and 6-224 with the same thickness but eachthin lossy film is formed from a different material. In someembodiments, both the material of the thin lossy films and the thicknessof the thin lossy films may be selected such that emission energy of adesired wavelength constructively interferes and is transmitted throughthe film.

FIG. 6-1 illustrates an embodiment where a combination of diffractiveelements and lenses are used to sort the emission energy by wavelength.A first layer 6-105 of the sensor chip may include a blazed phasegrating. The blazed grating may be blazed, for example, at an angle 4substantially equal to 40 degrees and the line spacing of the blazedgrating (A) may be substantially equal to 1.25 μm. One of skill in theart would appreciate that different blaze angles and periodicities maybe used to achieve separation of light of different wavelengths ofemission energy. Moreover, any suitable diffractive optical element maybe used to separate the different wavelengths of the emission energy.For example, a phase mask, an amplitude mask, a blazed grating or anoffset Fresnel lens may be used.

A second layer 6-106 of the sensor chip 2-260 may include one or moreFresnel lenses disposed beneath the first layer 6-105 to further sortand direct the emission energy to the sensors 6-107. Moreover, anysuitable lens element may be used to further separate the differentwavelengths of the emission energy. For example, a refractive lens maybe used instead of a Fresnel lens.

The Fresnel lens of the third lens layer and the blazed phase gratingmay be collectively referred to as diffractive optical elements (DOE).In some embodiments, the DOEs of have a thickness in the range ofapproximately 400 to 600 μm where width is in the range of approximately10 microns to 30 microns. An air spacer layer may be positioned directlyunder the DOEs and may have a thickness of approximately 150 m, in someembodiments. Alternatively, the spacer layer may have the same opticalthickness as the DOE focal length and/or be made of silicon dioxide.

The various components of FIG. 6-1 may be spaced apart at any suitabledistances. For example, the surface of the sensors may be located at adistance of 5 m beneath the Fresnel lens layer 6-106; the distance fromthe center of the lens 6-103 of the microscopy layer to the Fresnel lenslayer 6-106 may be 50.6 mm; the blazed phase grating 6-105 may belocated at a distance of approximately 100 m above the surface of thesensors. Alternatively, the distance from the bottom of the assay chipto the top of the grating 6-105 may be approximately 53 mm. The width ofthe sensor layer may be approximately 10 mm. The Fresnel lens may bespaced apart from each other within the layer by a distance ofapproximately 10 microns, approximately 20 microns, and approximately 30microns.

In some embodiments, an offset Fresnel lens may be used in which theFresnel lens has a center that may be offset from the center of thelens, such as lens 6-103 shown in FIG. 6-1. The center of the offsetFresnel lens, may be offset by approximately 50 microns, approximately60 microns, approximately 70 microns, or approximately 80 microns, butother distances are possible. In some embodiments, the offset Fresnellens may have a second offset which is the offset from the center of theoffset Fresnel lens to the center of the sensor, which may be, forexample, on the order of 10 um or 100 um, but other distances arepossible. The offset Fresnel lens may be created in any suitable way.For example, two sections, each with a different pitch of grating may beoverlaid to create a single offset Fresnel with improved efficiency overa binary offset Fresnel lens structure. In some embodiments, a “small”section with pitch 220 nm and a “big” section with pitch 440 nm may beoverlaid to create the offset Fresnel lens shown. The offset Fresnellens array may be positioned on top of the sensor array, may have alateral offset from the offset Fresnel lens element center to the sensorcenter, and may be designed to operate with a focal length in the rangeof approximately 80 microns to approximately 150 microns and designedfor a wavelength of 625 nm.

The layer directly under the imaging optics and the air spacer layer maybe a sensor layer. The sensor layer may include a plurality of sensors,including CMOS photo-sensitive sensors. The sensor layer may have anysuitable thickness, including in the range of approximately 6 microns toapproximately 8 microns. Alternatively, the sensor layer thickness maybe within the range of 2 um to 15 um. The sensors may be separated intoa plurality of segments or pixels and may have a width of approximately21 μm. The distance from the top of the sensor layer to a focal point ofthe emission energy may be approximately 30.3 mm.

The various layers of the assay chip and instrument need not be in theorder described above. In some embodiments, the focusing and/or sortingelements and the imaging optics of the instrument may be in reverseorder. For example, the blazed phase grating 6-105 may be placed afterthe Fresnel lens layer 6-106. Alternatively, the focusing and/or sortingelements and the imaging optics may be incorporated into a singlediffractive optical element (DOE). In addition, various components ofthe assay chip and instrument may be intermingled such that, forexample, imaging optics may occur both above and below the focusingand/or sorting elements.

Any of the interfaces between the layers, including the interfacebetween air and a layer of the system, described above in the system mayinclude an anti-reflection coating.

C. Optical Block of the Instrument

In some embodiments, the optical block of the instrument 1-120 mayinclude some or all of the optical components described above. Theoptical block may provide the optical components as arranged in FIGS.6-4A. FIG. 6-4A illustrates light having λ1 and λ2 passing through theoptical components shown in FIG. 6-4A. In addition to the componentsdescribed above, the optical block may include a first fiber connector6-401 where a first optical fiber carrying a first wavelength ofexcitation energy, λ1, may connect and a second fiber connector 6-402where a second optical fiber carrying a second wavelength of excitationenergy, λ2, may connect. By way of example and not limitation, the firstexcitation wavelength of the excitation energy may be 630-640 nm. Theoptical fiber connectors may be any suitable conventional connector,such as an FC or an LC connector. If two different wavelengths areinput, the wavelengths may be combined with a wavelength combiner 6-403,such as a dichroic or polychroic mirror. The second excitationwavelength may be 515-535 nm. The input excitation energy may be anysuitable polarization, such as linear polarization. In some embodiments,the fiber carrying the excitation energy may be apolarization-maintaining fiber. Optionally, excitation filters andpolarizers, such as optical fiber-to-free-space couplers, may be usedafter the optical fiber input to further filter or modifycharacteristics of the excitation energy.

The optical block may include one or more metal housings to hold lensesand other optical components for optical processing such as beamshaping. FIG. 6-4A illustrates four metal housings 6-405 through 6-408,each holding a lens and/or other optical components. There may be anynumber of lenses used to collimate and focus the excitation energy. Oneor more mirrors 6-411 and 6-412 are situated between some of the metalhousings for guiding the excitation energy towards the assay chip 2-110.In FIG. 6-4A, the first mirror 6-411 directs the excitation energy fromthe second housing 6-406 to the third housing 6-407 and the secondmirror 6-412 reflects the excitation energy from the fourth housing6-408 to a polychroic dielectric mirror 2-230. The polychroic dielectricmirror 2-230 directs the excitation energy towards an astigmatismcompensation filter 6-601.

In some embodiments, circularly polarized light may be directed into thesample well to cause the luminescent markers to emit luminescence withsimilar strength. A quarter-wave plate may be used to transfer thelinearly polarized light to circularly polarized light before it reachesthe assay chip. The polychroic dielectric mirror 2-230 directs theexcitation energy to the quarter wave plate 6-415. As illustrated inFIG. 6-4A, the quarter-wave plate 6-415 may be disposed between theastigmatism compensation filter 6-101 and the assay chip 2-110. Thecircularly polarized excitation energy is then directed towards theplurality of pixels on the assay chip. Excitation energy that is notdirected towards the pixels may be absorbed by a beam dump component6-417. Excitation energy that reaches the sample inside one or moresample wells will cause the sample to emit emission energy, which isdirected toward the sensor 2-260. The emission energy may pass throughoptical components such as polarization optics, the astigmatismcompensating element 6-101, the polychroic mirror 2-230 and a relay lens6-103. The polychroic mirror acts as a filer, which may be, by way ofexample, a notch filter, a spike filter or a cut-off filter. The relaylens 6-103 may image the emission energy toward the sensor. A portion ofthe emission energy may then pass through one or more emission filters6-421 and 6-422, situated above the sensor 2-260, which may furtherfilter the emission energy. In some embodiments, the emission filtersmay be tilted at an angle relative to the incident emission energypropagation direction in order to tune the transmission characteristicsof the filters and/or reduce interference caused by back reflections. Ifthe top filter 6-421 is tilted at an angle θ, the bottom filter 6-422may be tilted at the same angle θ, but about an axis that is orthogonalto that of the top filter's tilt, to ensure no astigmatism is introducedinto the emission radiation beam path.

FIG. 6-5 illustrates an example of a ray trace that represents anoptical path through the apparatus from the assay chip 2-110 to thesensor chip 2-260. Each starting point in assay chip 2-110 represents apixel that emits emission energy and is imaged by the system to acorresponding pixel on sensor chip 2-260. The path may exists along adistance of approximately 55.4 mm but any suitable distance may be used.In the example illustrated, rays emitted from the sample well array arefiltered and focused toward the sensor using the optical elementsdescribed above. FIG. 6-5 illustrates one possible relay lens 6-103 thatcomprises six individual lenses. It should be recognized that any numberof lens and/or other optical elements may be used as a relay lens.

IV. SENSORS

The present disclosure provides various embodiments of sensors, sensoroperation, and signal processing methods. According to some embodiments,a sensor 2-122 at a pixel of the sensor chip 2-260 may comprise anysuitable sensor capable of receiving emission energy from one or moretags in the sample well, and producing one or more electrical signalsrepresentative of the received emission energy. In some embodiments, asensor may comprise at least one a photodetector (e.g., a p-n junctionformed in a semiconductor substrate). FIG. 7-1A and FIG. 7-1B depictsone embodiment of a sensor that may be fabricated within a pixel 2-100of a sensor chip.

According to some embodiments, a sensor 2-122 may be formed at eachpixel 2-100 of a sensor chip. The sensor may be associated with a samplewell 2-211 of the assay chip. There may be one or more transparentlayers 7-110 above the sensor, so that emission from the sample well maytravel to the sensor without significant attenuation. The sensor 2-122may be formed in a semiconductor substrate 7-120 at a base of the pixel,according to some embodiments, and be located on a same side of thesample well as the assay chip (not shown).

The sensor may comprise one or more semiconductor junction photodetectorsegments. Each semiconductor junction may comprise a well of a firstconductivity type. For example, each semiconductor junction may comprisean n-type well formed in a p-type substrate, as depicted in the drawing.According to some embodiments, a sensor 2-122 may be arranged as abulls-eye detector 7-162, as depicted in the plan view of FIG. 7-1B. Afirst photodetector 7-124 may be located at a center of the sensor, anda second annular photodetector 7-122 may surround the centerphotodetector. Electrical contacts to the wells may be made throughconductive traces 7-134 formed at a first or subsequent metallizationlevel and through conductive vias 7-132. There may be a region of highlydoped semiconductor material 7-126 at contact regions of the vias. Insome embodiments, a field oxide 7-115 may be formed at surfaces betweenthe photodetectors and may cover a portion of each photodetector. Insome implementations, there may be additional semiconductor devices7-125 (e.g., transistors, amplifiers, etc.) formed within the pixeladjacent to the sensor 2-122. There may be additional metallizationlevels 7-138, 7-136 within the pixel.

In some implementations, a metallization level 7-136 may extend across amajority of the pixel and have an opening centered above thephotodetector 7-124, so that emission from the sample well can reach thesensor. In some cases, a metallization level 7-136 may serve as areference potential or a ground plane, and additionally serve as anoptical block to prevent at least some background radiation (e.g.,radiation from an excitation source or from the ambient environment)from reaching the sensor 2-260.

As depicted in FIG. 7-1A and FIG. 7-1B, a sensor 2-122 may be subdividedinto a plurality of photodetector segments 7-122, 7-124 that arespatially and electrically separated from each other. In someembodiments, segments of a sensor 2-122 may comprise regions ofoppositely-doped semiconductor material. For example, a first chargeaccumulation well 7-124 for a first sensor segment may be formed bydoping a first region of a substrate to have a first conductivity type(e.g., n-type) within the first well. The substrate may be p-type. Asecond charge accumulation well 7-122 for a second sensor segment may beformed by doping a second region of the substrate to have the firstconductivity type within the second well. The first and second wells maybe separated by a p-type region of the substrate.

The plurality of segments of the sensor 2-122 may be arranged in anysuitable way other than a bulls-eye layout, and there may be more thantwo segments in a sensor. For example, in some embodiments, a pluralityof photodetector segments 7-142 may be laterally separated from oneanother to form a stripe sensor 7-164, as depicted in FIG. 7-1C. In someembodiments, a quad (or quadrant) sensor 7-166 may be formed byarranging the segments 7-144 in a quad pattern, as depicted in FIG.7-1D. In some implementations, arc segments 7-146 may be formed incombination with a bulls-eye pattern, as depicted in FIG. 7-1E, to forman arc-segmented sensor 7-168. Another sensor configuration may comprisepie-piece sections, which may include individual sensors arranged inseparate section of a circle. In some cases, sensor segments may bearranged symmetrically around a sample well 2-211 or asymmetricallyaround a sample well. The arrangement of sensor segments is not limitedto only the foregoing arrangements, and any suitable distribution ofsensor segments may be used.

The inventors have found that a quadrant sensor 7-166, pie-sectorsensor, or similar sector sensor can scale to smaller pixel sizes morefavorably than other sensor configurations. Quadrant and sectordetectors may consume less pixel area for a number of wavelengthsdetected and active sensor area.

Sensors may be arranged in various geometric configurations. In someexamples, sensors are arranged in a square configurations or hexagonalconfiguration.

Sensors may be sized and positioned in any suitable way to capture theemitted emission energy from a sample well. For example, the sensor maybe centered underneath the sample well and have planar dimensions of 5um×5 um. Alternatively, each sub-sensor may have dimensions of 1.6 um×10um with a pitch of 4.6 um (i.e. 3 um gap in between each sub-sensor).

Sensors of the present disclosure may be independently (or individually)addressable. An individually addressable is capable of detecting asignal and providing an output independent of other sensors. Anindividually addressable sensor may be individually readable.

In some embodiments, a stacked sensor 7-169 may be formed by fabricatinga plurality of separated sensor segments 7-148 in a vertical stack, asdepicted in FIG. 7-1F. For example, the segments may be located oneabove the other, and there may, or may not, be insulating layers betweenthe stacked segments. Each vertical layer may be configured to absorbemission energy of a particular energy, and pass emission at differentenergies. For example, a first detector may absorb and detectshorter-wavelength radiation (e.g., blue-wavelength radiation belowabout 500 nm from a sample). The first detector may pass green- andred-wavelength emissions from a sample. A second detector may absorb anddetect green-wavelength radiation (e.g., between about 500 nm and about600 nm) and pass red emissions. A third detector may absorb and detectthe red emissions. Reflective films 7-149 may be incorporated in thestack, in some embodiments, to reflect light of a selected wavelengthband back through a segment. For example, a film may reflectgreen-wavelength radiation that has not been absorbed by the secondsegment back through the second segment to increase its detectionefficiency.

In some embodiments with vertically-stacked sensor segments,emission-coupling components may not be included at the sample well toproduce distinct spatial distribution patterns of sample emission thatare dependent on emission wavelength. Discernment of spectrallydifferent emissions may be achieved with a vertically-stacked sensor7-169 by analyzing the ratio of signals from its stacked segment,according to some embodiments.

In some embodiments, segments of a sensor 2-122 are formed from silicon,though any suitable semiconductor (e.g., Ge, GaAs, SiGe, InP, etc.) maybe used. In some embodiments, a sensor segment may comprise an organicphotoconductive film. In other embodiments, quantum dot photodetectorsmay be used for sensor segments. Quantum dot photodetectors may respondto different emission energies based on the size of the quantum dot. Insome embodiments, a plurality of quantum dots of varying sizes may beused to discriminate between different emission energies or wavelengthsreceived from the sample well. For example, a first segment may beformed from quantum dots having a first size, and a second segment maybe formed from quantum dots having a second size. In variousembodiments, sensors 2-122 may be formed using conventional CMOSprocesses.

As described above, emission-coupling components may be fabricatedadjacent the sample well in some embodiments. The sorting elements 2-243can alter emission from a sample within the sample well 2-211 to producedistinct spatial distribution patterns of sample emission that aredependent on emission wavelength. FIG. 7-2A depicts an example of afirst spatial distribution pattern 7-250 that may be produced from afirst sample at a first wavelength. The first spatial distributionpattern 7-250 may have a prominent central lobe directed toward acentral segment of a bulls-eye sensor 7-162, for example, as shown inFIG. 7-2B. Such a pattern 7-250 may be produced by any suitablediffractive element when the sample emits at a wavelength of about 663nm. A projected pattern 7-252 incident on the sensor may appear asillustrated in FIG. 7-2B.

FIG. 7-2C depicts a spatial distribution pattern 7-260 that may beproduced from a second sample emitting at a second wavelength from thesame sample well, according to some embodiments. The second spatialdistribution pattern 7-260 may comprise two lobes of radiation anddiffer from the first spatial distribution pattern 7-250. A projectedpattern 7-262 of the second spatial distribution pattern 7-260 mayappear as depicted in FIG. 7-2D, according to some embodiments. Thesecond spatial distribution pattern 7-260 may be produced by anysuitable diffractive element when the sample emits at a wavelength ofabout 687 nm.

The segments of a sensor 2-122 may be arranged to detect particularemission energies, according to some embodiments. For example,emission-coupling structures adjacent the sample well and segments of asensor may be designed in combination to increase signal differentiationbetween particular emission energies. The emission energies maycorrespond to selected tags that will be used with the sensor chip. Asan example, a bulls-eye sensor 7-162 could have its segments sizedand/or located to better match the projected patterns 7-260, 7-262 froma sample, so that regions of higher intensity fall more centrally withinactive segments of the sensor. Alternatively or additionally,diffractive elements may be designed to alter the projected patterns7-260, 7-262 so that intense regions fall more centrally within segmentsof the sensor.

Although a sensor 2-122 may comprise two segments, it is possible insome embodiments to discern more than two spectrally-distinct emissionbands from a sample. For example, each emission band may produce adistinct projected pattern on the sensor segments and yield a distinctcombination of signals from the sensor segments. The combination ofsignals may be analyzed to discern and identify the emission band. FIG.7-2E through FIG. 7-2H represent results from numerical simulations ofsignals from a two-segment sensor 2-122 exposed to four distinctemission patterns. As can be seen, each combination of signals from thetwo sensor segments is distinct, and can be used to discriminate betweenemitters at the four wavelengths. For the simulation, because the outerdetector segment of the bulls-eye sensor 7-162 had a larger area, moresignal was integrated for that detector. Additionally, light thatimpinged on an area between the detectors generated carriers that maydrift towards either detector segment and contribute to signals fromboth segments.

In some embodiments, there may be N photodetector segments per pixel,where N may be any integer value. In some embodiments, N may be greaterthan or equal to 1 and less than or equal to 10. In other embodiments, Nmay be greater than or equal to 2 and less than or equal to 5. Thenumber M of discernible sample emissions (e.g., distinct emissionwavelengths from different luminescent tags) that may be detected by theN detectors may be equal to or greater than N. The discernment of Msample emissions may be achieved by evaluating the ratio of signals fromeach sensor segment, according to some embodiments. In someimplementations, the ratio, sum and/or amplitudes of the receivedsignals may be measured and analyzed to determine a characteristicwavelength of emission from the sample well.

In some embodiments, more than one emitter may emit at differentcharacteristic wavelengths in a given time window within a sample well2-211. A sensor 2-122 may simultaneously detect signals from multipleemissions at different wavelengths and provide the summed signal fordata processing. In some implementations, multi-wavelength emission maybe distinguishable as another set of signal values from the sensorsegments (e.g., signal values different from those shown in FIG. 7-2Ethrough FIG. 7-2H). The signal values may be analyzed to discern thatmulti-wavelength emission has occurred and to identify a particularcombination of emitters associated with the emissions.

The inventors have also contemplated and analyzed a bulls-eye sensorhaving four concentric segments. Signals from the segments are plottedin FIG. 7-2I and FIG. 7-2J for the same emission conditions associatedwith FIG. 7-2G and FIG. 7-2H, respectively. The four-segment bulls-eyesensor also shows discernable signals that may be analyzed to identify aparticular emitter within the sample well.

When wavelength filtering is used at each sensor segment, or thespectral separation is high, each segment of a sensor may detectsubstantially only a selected emission band. For example, a firstwavelength may be detected by a first segment, a second wavelength maybe detected by a second segment, and a third wavelength may be detectedby a third segment.

Referring again to FIG. 7-1A, there may be additional electroniccircuitry 7-125 within a pixel 2-100 that may be used to collect andreadout signals from each segment of a sensor 2-122. FIG. 7-3A and FIG.7-3D depict circuitry that may be used in combination with amulti-segment sensor, according to some embodiments. As an example,signal collection circuitry 7-310 may comprise three transistors foreach sensor segment. An arrangement of the three transistors is depictedin FIG. 7-3B, according to some implementations. A signal level at acharge accumulation node 7-311 associated with each segment may be resetby a reset transistor RST, and a signal level for the segment(determined by the amount of charge at the charge accumulation node) maybe read out with a read transistor RD.

The pixel circuitry may further include amplification and correlateddouble-sampling circuitry 7-320, according to some embodiments. Theamplification and double-sampling circuitry may comprise transistorsconfigured to amplify signals from the sensor segments as well astransistors configured to reset the voltage level at thecharge-accumulation node and to read a background, or “reset”, signal atthe node when no emission energy is present on the sensor (e.g., priorto application of excitation energy at the sample well) and to read asubsequent emission signal, for example.

According to some embodiments, correlated double sampling is employed toreduce background noise by subtracting a background or reset signallevel from the detected emission signal level. The collected emissionsignal and background signal associated with each segment of the sensormay be read out onto column lines 7-330. In some embodiments, anemission signal level and background signal are time-multiplexed onto acommon column line. There may be a separate column line for each sensorsegment. Signals from the column lines may be buffered and/or amplifiedwith amplification circuitry 7-340 (which may be located outside of anactive pixel array), and provided for further processing and analysis.In some embodiments the subtraction of the double-sampled signals iscalculated off-chip, e.g., by a system processor. In other embodiments,the subtraction may be performed on chip or in circuitry of theinstrument.

Some embodiments of correlated double sampling may operate by selectinga row to sample, wherein the sensors associated with the row haveintegrated signal charges over a sampling period and contain signallevels. The signal levels may be simultaneously read out onto thecolumns lines. After sampling the integrated signal levels, all thepixels in the selected row may be reset and immediately sampled. Thisreset level may be correlated to the next integrated signal that startsaccumulating after the reset is released, and finishes integrating aframe time later when the same row is selected again. In someembodiments, the reset values of the frame may be stored off-chip sothat when the signals have finished integrating and have been sampled,the stored correlated reset values can be subtracted.

In some embodiments, a sensor 2-122 with more than two segments mayrequire additional circuitry. FIG. 7-3C depicts signal-collection 7-312,amplification 7-320, and double-sampling circuitry associated with aquad sensor. According to some embodiments, signals from two or moresegments may be time-multiplexed onto a common signal channel at thepixel, as depicted in the drawing. The time-multiplexed signals mayinclude sampled background signals for each segment for noisecancellation. Additionally, the signals from two or more segments may betime-multiplexed onto a common column line.

According to some embodiments, temporal signal-acquisition techniquesmay be used to reduce background signal levels from an excitation sourceor sources, and/or discern different emissions from different emittersassociated with a sample. FIG. 7-4A depicts fluorescent emission anddecay from two different emitters that may be used to tag a sample,according to some embodiments. The two emissions have appreciablydifferent time-decay characteristics. A first time-decay curve 7-410from a first emitter may correspond to a common fluorescent moleculesuch as rhodamine. A second time-decay curve 7-420 may be characteristicof a second emitter, such as a quantum dot or a phosphorescent emitter.Both emitters exhibit an emission-decay tail that extends for some timeafter initial excitation of the emitter. In some embodiments,signal-collection techniques applied during the emission-decay tail maybe timed to reduce a background signal from an excitation source, insome embodiments, and to distinguish between the emitters, in someembodiments.

According to some implementations, time-delayed sampling may be employedduring the emission-decay tail to reduce a background signal due toradiation from an excitation source. FIG. 7-4B and FIG. 7-4C illustratetime-delay sampling, according to some embodiments. FIG. 7-4B depictsthe temporal evolution of an excitation pulse 7-440 of excitation energyfrom an excitation source, and a subsequent emission pulse 7-450 thatmay follow from a sample that is excited within the sample well. Theexcitation pulse 7-440 may result from driving the excitation sourcewith a drive signal 7-442 for a brief period of time, as depicted inFIG. 7-4C. For example, the drive signal may begin at a first time t₁and end at a second time t₂. The duration of the drive signal (t₂−t₁)may be between about 1 picosecond and about 50 nanoseconds, according tosome embodiments, though shorter durations may be used in someimplementations.

At a time t₃ following termination of the drive signal for theexcitation source, a sensor 2-260 (or sensor segment) at the pixel maybe gated to accumulate charge at a charge accumulation node 7-311 duringa second time interval 7-452 extending from a time t₃ to a time t₄. Thesecond time interval may be between about 1 nanosecond and about 50microseconds, according to some embodiments, though other durations maybe used in some implementations. As can be seen in reference to FIG.7-4B, a charge accumulation node will collect more signal charges due tothe emitting sample then due to the excitation source. Accordingly, animproved signal-to-noise ratio may be obtained.

Referring again to FIG. 7-4A, because of the different temporal emissioncharacteristics of the emitters, corresponding signals at a sensor maypeak at different times. In some implementations, signal-acquisitiontechniques applied during the emission-decay tail may be used to discerndifferent emitters. In some embodiments, temporal detection techniquesmay be used in combination with spatial and spectral techniques (asdescribed above in connection with FIG. 7-2, for example) to discerndifferent emitters.

FIG. 7-4D through FIG. 7-4H illustrate how double-sampling at a sensor,or sensor segment, can be used to distinguish between two emittershaving different temporal emission characteristics. FIG. 7-4D depictsemission curves 7-470, 7-475 associated with a first emitter and secondemitter, respectively. As an example, the first emitter may be a commonfluorophore such as rhodamine, and the second emitter may be a quantumdot or phosphorescent emitter.

FIG. 7-4E represents dynamic voltage levels at a charge accumulationnode 7-311 that may occur in response to the two different emissioncharacteristics of FIG. 7-4D. In the example, a first voltage curve7-472 corresponding to the fluorescent emitter may change more rapidly,because of the shorter emission span, and reach its maximum (or minimum,depending on the polarity of the node) at a first time t₁. The secondvoltage curve 7-477 may change more slowly due to the longer emissioncharacteristics of the second emitter, and reach its maximum (orminimum) at a second time t₂.

In some embodiments, sampling of the charge-accumulation node may bedone at two times t₃, t₄ after the sample excitation, as depicted inFIG. 7-4F. For example, a first read signal 7-481 may be applied to readout a first voltage value from the charge-accumulation node at a firsttime t₃. Subsequently, a second read signal 7-482 may be applied to readout a second voltage value from the charge-accumulation node at a secondtime t₄ without resetting the charge-accumulation node between the firstread and second read. An analysis of the two sampled signal values maythen be used to identify which of the two emitters provided the detectedsignal levels.

FIG. 7-4G depicts an example of two signals from the first read andsecond read that may be obtained for the first emitter having anemission curve 7-470 as depicted in FIG. 7-4D. FIG. 7-4H depicts anexample of two signals from the first read and second read that may beobtained for the second emitter having an emission curve 7-475 asdepicted in FIG. 7-4D. For example the sampling sequence shown in FIG.7-4F for the first emitter will sample the curve 7-472 and obtainapproximately the same values at the two read times. In the case of thesecond emitter, the sampling sequence depicted in FIG. 7-4F samples twodifferent values of the curve 7-477 at the two read times. The resultingpairs of signals from the two read times distinguish between the twoemitters, and can be analyzed to identify each emitter. According tosome embodiments, double sampling for background subtraction may also beexecuted to subtract a background signal from the first and second readsignals.

In operation, sensors 2-260 of a sensor chip may be subjected to awavelength calibration procedure prior to data collection from aspecimen to be analyzed. The wavelength calibration procedure mayinclude subjecting the sensors to different known energies havingcharacteristic wavelengths that may, or may not, correspond tofluorophore wavelengths that may be used with a sensor chip. Thedifferent energies may be applied in a sequence so calibration signalscan be recorded from the sensors for each energy. The calibrationsignals may then be stored as reference signals, that may be used toprocess real data acquisition and to determine what emission wavelengthor wavelengths are detected by the sensors.

Any suitable sensor capable of acquiring time bin information may beused for measurements to detect lifetimes of luminescent markers. Thesensors are aligned such that each sample well has at least one sensorregion to detect luminescence from the sample well. In some embodiments,the integrated device may include Geiger mode avalanche photodiodearrays and/or single photon avalanche diode arrays (SPADs). The sensormay include IR-enhanced CMOS sensors which may include Si—Ge materialsand/or modified layers such as “black silicon”. These materials mayenable the sensor to detect luminescent markers that emit in theinfrared, and/or are otherwise poorly detected by non-IR-enhanced CMOSsensors.

Described herein is an integrated photodetector that can accuratelymeasure, or “time-bin,” the timing of arrival of incident photons, andwhich may be used in a variety of applications, such as sequencing ofnucleic acids (e.g., DNA sequencing), for example. In some embodiments,the integrated photodetector can measure the arrival of photons withnanosecond or picosecond resolution, which can facilitate time-domainanalysis of the arrival of incident photons.

Some embodiments relate to an integrated circuit having a photodetectorthat produces charge carriers in response to incident photons and whichis capable of discriminating the timing at which the charge carriers aregenerated by the arrival of incident photons with respect to a referencetime (e.g., a trigger event). In some embodiments, a charge carriersegregation structure segregates charge carriers generated at differenttimes and directs the charge carriers into one or more charge carrierstorage regions (termed “bins”) that aggregate charge carriers producedwithin different time periods. Each bin stores charge carriers producedwithin a selected time interval. Reading out the charge stored in eachbin can provide information about the number of photons that arrivedwithin each time interval. Such an integrated circuit can be used in anyof a variety of applications, such as those described herein.

An example of an integrated circuit having a photodetection region and acharge carrier segregation structure will be described. In someembodiments, the integrated circuit may include an array of pixels, andeach pixel may include one or more photodetection regions and one ormore charge carrier segregation structures, as discussed below.

FIG. 7-5 shows a diagram of a pixel 100, according to some embodiments.Pixel 100 includes a photon absorption/carrier generation region 102(also referred to as a photodetection region), a carrier travel/captureregion 106, a carrier storage region 108 having one or more chargecarrier storage regions, also referred to herein as “charge carrierstorage bins” or simply “bins,” and readout circuitry 110 for readingout signals from the charge carrier storage bins.

The photon absorption/carrier generation region 102 may be a region ofsemiconductor material (e.g., silicon) that can convert incident photonsinto photogenerated charge carriers. The photon absorption/carriergeneration region 102 may be exposed to light, and may receive incidentphotons. When a photon is absorbed by the photon absorption/carriergeneration region 102 it may generate photogenerated charge carriers,such as an electron/hole pair. Photogenerated charge carriers are alsoreferred to herein simply as “charge carriers.”

An electric field may be established in the photon absorption/carriergeneration region 102. In some embodiments, the electric field may be“static,” as distinguished from the changing electric field in thecarrier travel/capture region 106. The electric field in the photonabsorption/carrier generation region 102 may include a lateralcomponent, a vertical component, or both a lateral and a verticalcomponent. The lateral component of the electric field may be in thedownward direction of FIG. 7-5, as indicated by the arrows, whichinduces a force on photogenerated charge carriers that drives themtoward the carrier travel/capture region 106. The electric field may beformed in a variety of ways.

In some embodiments, one or more electrodes may be formed over thephoton absorption/carrier generation region 102. The electrodes(s) mayhave voltages applied thereto to establish an electric field in thephoton absorption/carrier generation region 102. Such electrode(s) maybe termed “photogate(s).” In some embodiments, photon absorption/carriergeneration region 102 may be a region of silicon that is fully depletedof charge carriers.

In some embodiments, the electric field in the photon absorption/carriergeneration region 102 may be established by a junction, such as a PNjunction. The semiconductor material of the photon absorption/carriergeneration region 102 may be doped to form the PN junction with anorientation and/or shape that produces an electric field that induces aforce on photogenerated charge carriers that drives them toward thecarrier travel/capture region 106. In some embodiments, the P terminalof the PN junction diode may connected to a terminal that sets itsvoltage. Such a diode may be referred to as a “pinned” photodiode. Apinned photodiode may promote carrier recombination at the surface, dueto the terminal that sets its voltage and attracts carriers, which canreduce dark current. Photogenerated charge carriers that are desired tobe captured may pass underneath the recombination area at the surface.In some embodiments, the lateral electric field may be established usinga graded doping concentration in the semiconductor material.

As illustrated in FIG. 7-5, a photon may be captured and a chargecarrier 101A (e.g., an electron) may be produced at time t1. In someembodiments, an electrical potential gradient may be established alongthe photon absorption/carrier generation region 102 and the carriertravel/capture region 106 that causes the charge carrier 101A to travelin the downward direction of FIG. 7-5 (as illustrated by the arrowsshown in FIG. 7-5). In response to the potential gradient, the chargecarrier 101A may move from its position at time t1 to a second positionat time t2, a third position at time t3, a fourth position at time t4,and a fifth position at time t5. The charge carrier 101A thus moves intothe carrier travel/capture region 106 in response to the potentialgradient.

The carrier travel/capture region 106 may be a semiconductor region. Insome embodiments, the carrier travel/capture region 106 may be asemiconductor region of the same material as photon absorption/carriergeneration region 102 (e.g., silicon) with the exception that carriertravel/capture region 106 may be shielded from incident light (e.g., byan overlying opaque material, such as a metal layer).

In some embodiments, and as discussed further below, a potentialgradient may be established in the photon absorption/carrier generationregion 102 and the carrier travel/capture region 106 by electrodespositioned above these regions. However, the techniques described hereinare not limited as to particular positions of electrodes used forproducing an electric potential gradient. Nor are the techniquesdescribed herein limited to establishing an electric potential gradientusing electrodes. In some embodiments, an electric potential gradientmay be established using a spatially graded doping profile. Any suitabletechnique may be used for establishing an electric potential gradientthat causes charge carriers to travel along the photonabsorption/carrier generation region 102 and carrier travel/captureregion 106.

A charge carrier segregation structure may be formed in the pixel toenable segregating charge carriers produced at different times. In someembodiments, at least a portion of the charge carrier segregationstructure may be formed over the carrier travel/capture region 106. Aswill be described below, the charge carrier segregation structure mayinclude one or more electrodes formed over the carrier travel/captureregion 106, the voltage of which may be controlled by control circuitryto change the electric potential in the carrier travel/capture region106.

The electric potential in the carrier travel/capture region 106 may bechanged to enable capturing a charge carrier. The potential gradient maybe changed by changing the voltage on one or more electrodes overlyingthe carrier travel/capture region 106 to produce a potential barrierthat can confine a carrier within a predetermined spatial region. Forexample, the voltage on an electrode overlying the dashed line in thecarrier travel/capture region 106 of FIG. 7-5 may be changed at time t5to raise a potential barrier along the dashed line in the carriertravel/capture region 106 of FIG. 7-5, thereby capturing charge carrier101A. As shown in FIG. 7-5, the carrier captured at time t5 may betransferred to a bin “bin0” of carrier storage region 108. The transferof the carrier to the charge carrier storage bin may be performed bychanging the potential in the carrier travel/capture region 106 and/orcarrier storage region 108 (e.g., by changing the voltage ofelectrode(s) overlying these regions) to cause the carrier to travelinto the charge carrier storage bin.

Changing the potential at a certain point in time within a predeterminedspatial region of the carrier travel/capture region 106 may enabletrapping a carrier that was generated by photon absorption that occurredwithin a specific time interval. By trapping photogenerated chargecarriers at different times and/or locations, the times at which thecharge carriers were generated by photon absorption may bediscriminated. In this sense, a charge carrier may be “time binned” bytrapping the charge carrier at a certain point in time and/or spaceafter the occurrence of a trigger event. The time binning of a chargecarrier within a particular bin provides information about the time atwhich the photogenerated charge carrier was generated by absorption ofan incident photon, and thus likewise “time bins,” with respect to thetrigger event, the arrival of the incident photon that produced thephotogenerated charge carrier.

FIG. 7-6 illustrates capturing a charge carrier at a different point intime and space. As shown in FIG. 7-6, the voltage on an electrodeoverlying the dashed line in the carrier travel/capture region 106 maybe changed at time t9 to raise a potential barrier along the dashed linein the carrier travel/capture region 106 of FIG. 7-6, thereby capturingcarrier 101B. As shown in FIG. 7-6, the carrier captured at time t9 maybe transferred to a bin “bin1” of carrier storage region 108. Sincecharge carrier 101B is trapped at time t9, it represents a photonabsorption event that occurred at a different time (i.e., time t6) thanthe photon absorption event (i.e., at τ1) for carrier 101A, which iscaptured at time t5.

Performing multiple measurements and aggregating charge carriers in thecharge carrier storage bins of carrier storage region 108 based on thetimes at which the charge carriers are captured can provide informationabout the times at which photons are captured in the photonabsorption/carrier generation area 102. Such information can be usefulin a variety of applications, as discussed above.

In some embodiments, the duration of time each time bin captures afteran excitation pulse may vary. For example, shorter time bins may be usedto detect luminescence shortly after the excitation pulse, while longertime bins may be used at times further from an excitation pulse. Byvarying the time bin intervals, the signal to noise ratio formeasurements of the electrical signal associated with each time bin maybe improved for a given sensor. Since the probability of a photonemission event is higher shortly after an excitation pulse, a time binwithin this time may have a shorter time interval to account for thepotential of more photons to detect. While at longer times, theprobability of photon emission may be less and a time bin detectingwithin this time may be longer to account for a potential fewer numberof photons. In some embodiments, a time bin with a significantly longertime duration may be used to distinguish among multiple lifetimes. Forexample, the majority of time bins may capture a time interval in therange of approximately 0.1-0.5 ns, while a time bin may capture a timeinterval in the range of approximately 2-5 ns. The number of time binsand/or the time interval of each bin may depend on the sensor used todetect the photons emitted from the sample object. Determining the timeinterval for each bin may include identifying the time intervals neededfor the number of time bins provided by the sensor to distinguish amongluminescent markers used for analysis of a sample. The distribution ofthe recorded histogram may be compared to known histograms of markersunder similar conditions and time bins to identify the type of marker inthe sample well. Different embodiments of the present application maymeasure lifetimes of markers but vary in the excitation energies used toexcite a marker, the number of sensor regions in each pixel, and/or thewavelength detected by the sensors.

V. EXCITATION SOURCES

The excitation source 2-250 may be any suitable source that is arrangedto deliver excitation energy to at least one sample well 2-111 of theassay chip. Pixels on the assay chip may be passive source pixels. Theterm “passive source pixel” is used to refer to a pixel wherein theexcitation energy is delivered to the pixel from a region outside thepixel or pixel array of the assay chip, e.g., the excitation may be inthe instrument.

According to some embodiments, an excitation source may excite a samplevia a radiative process. For example, an excitation source may providevisible radiation (e.g., radiation having a wavelength between about 350nm and about 750 nm), near-infrared radiation (e.g., radiation having awavelength between about 0.75 micron and about 1.4 microns), and/orshort wavelength infrared radiation (e.g., radiation having a wavelengthbetween about 1.4 microns and about 3 microns) to at least oneexcitation region 3-215 of at least one sample well of the assay chip.In some embodiments, a radiative excitation source may provide energy toexcite an intermediary (e.g., a molecule, a quantum dot, or a layer ofmaterial comprising selected molecules and/or quantum dots) that isimmediately adjacent an excitation region of a sample well. Theintermediary may transfer its energy to a sample via a non-radiativeprocess (e.g., via FRET or DET).

In some embodiments, an excitation source may provide more than onesource of excitation energy. For example, a radiative excitation sourcemay deliver excitation energies having two or more distinct spectralcharacteristics. As an example, a multi-color LED may emit energiescentered at two or more wavelengths, and these energies may be deliveredto an excitation region of a sample well.

In overview and according to some embodiments, an instrument may includeat least one excitation source 2-250 to provide excitation energy to atleast one excitation region of at least one sample well of the assaychip or to at least one intermediary that converts or couples theexcitation energy to at least one sample within one or more excitationregions. As depicted in FIG. 2-3, radiation excitation energy 2-251 froman excitation source 2-250 may impinge on a region around a sample well2-211, for example. In some embodiments, there may be excitationcoupling structures 2-223 that aid in concentrating the incidentexcitation energy within an excitation region 2-215 of the sample well.

An excitation source may be characterized by one or more distinctspectral bands each having a characteristic wavelength. Forinstructional purposes only, an example of spectral emission from anexcitation source is depicted in spectral graph of FIG. 8-1A. Theexcitation energy may be substantially contained within a spectralexcitation band 8-110. A peak wavelength 8-120 of the spectralexcitation band may be used to characterize the excitation energy. Theexcitation energy may also be characterized by a spectral distribution,e.g., a full-width-half-maximum (FWHM) value as shown in the drawing. Anexcitation source producing energy as depicted in FIG. 8-1A, may becharacterized as delivering energy at a wavelength of approximately 540nm radiation and having a FWHM bandwidth of approximately 55 nm.

FIG. 8-1B depicts spectral characteristics of an excitation source (orexcitation sources) that can provide two excitation energy bands to oneor more sample wells. According to some embodiments, a first excitationband 8-112 is at approximately 532 nm, and a second excitation band8-114 is at approximately 638 nm, as illustrated in the drawing. In someembodiments, a first excitation band may be at approximately 638 nm, anda second excitation band may be at approximately 650 nm. In someembodiments, a first excitation band may be at approximately 680 nm, anda second excitation band may be at approximately 690 nm. According tosome embodiments, the peaks of the excitation bands may be within ±5 nmof these values.

In some cases, a radiative excitation source may produce a broadexcitation band as depicted in FIG. 8-1A. A broad excitation band 8-110may have a bandwidth greater than approximately 20 nm, according to someembodiments. A broad excitation band may be produced by a light emittingdiode (LED), for example. In some implementations, a radiativeexcitation source may produce a narrow excitation band, as depicted inFIG. 8-1B. A narrow excitation band may be produced by a laser diode,for example, or may be produced by spectrally filtering an output froman LED.

In some embodiments, the excitation source may be a light source. Anysuitable light source may be used. Some embodiments may use incoherentsources and other embodiments may use coherent light sources. By way ofexample and not limitation, incoherent light sources according to someembodiments may include different types of light emitting diodes (LEDs)such as organic LEDs (OLEDs), quantum dots (QLEDs), nanowire LEDs, and(in)organic semiconductor LEDs. By way of example and not limitation,coherent light sources according to some embodiments may includedifferent types of lasers such as organic lasers, quantum dot lasers,vertical cavity surface emitting lasers (VCSELs), edge emitting lasers,and distributed-feedback (DFB) laser diodes. Additionally oralternatively, slab-coupled optical waveguide laser (SCOWLs) or otherasymmetric single-mode waveguide structures may be used. Additionally oralternatively, a solid state laser such as Nd:YAG or Nd:Glass, pumped bylaser diodes or flashlamps, may be used. Additionally or alternatively,a laser-diode-pumped fiber laser may be used. In some embodiments, theoutput of a laser excitation source may be doubled in frequency to halfthe wavelength, in a nonlinear crystal, or a Periodically Poled LithiumNiobate (PPLN) or other similar periodically poled nonlinear crystal.This frequency doubling process may allow use of efficient lasers togenerate wavelengths more suitable for excitation. There may be morethan one type of excitation source for an array of pixels. In someembodiments, different types of excitation sources may be combined. Theexcitation source may be fabricated according to conventionaltechnologies that are used to fabricate a selected type of excitationsource.

The characteristic wavelength of a source of excitation energy may beselected based upon a choice of luminescent markers that are used in theassay analysis. In some implementations, the characteristic wavelengthof a source of excitation energy is selected for direct excitation(e.g., single photon excitation) of a chosen fluorophore. In someimplementations, the characteristic wavelength of a source of excitationenergy is selected for indirect excitation (e.g., multi-photonexcitation or harmonic conversion to a wavelength that will providedirect excitation). In some embodiments, excitation energy may begenerated by a light source that is configured to generate excitationenergy at a particular wavelength for application to a sample well. Insome embodiments, a characteristic wavelength of the excitation sourcemay be less than a characteristic wavelength of corresponding emissionfrom the sample. In some implementations, a characteristic wavelength ofthe excitation source may be greater than a characteristic wavelength ofemission from the sample, and excitation of the sample may occur throughmulti-photon absorption.

The excitation source may include a battery or any other power supply,which may be located somewhere other than the integrated bioanalysisdevice. For example, the excitation source may be located in aninstrument and the power may be coupled to the integrated bioanalysisdevice via conducting wires and connectors.

According to some embodiments, one or more excitation sources may belocated external to the integrated device, and may be arranged todeliver pulses of light to an integrated device having sample wells. Thepulses of light may be coupled to a plurality of sample wells and usedto excite one or more markers within the wells, for example. The one ormore excitation sources may deliver pulses of light at one or morecharacteristic wavelengths, according to some implementations. In somecases, an excitation source may be packaged as an exchangeable modulethat mounts in or couples to a base instrument, into which theintegrated device may be loaded. Energy from an excitation source may bedelivered radiatively or non-radiatively to at least one sample well orto at least one sample in at least one sample well. In someimplementations, an excitation source having a controllable intensitymay be arranged to deliver excitation energy to a plurality of pixels ofan integrated device. The pixels may be arranged in a linear array(e.g., row or column), or in a 2D array (e.g., a sub-area of the arrayof pixels or the full array of pixels).

Any suitable light source may be used for an excitation source. Someembodiments may use incoherent light sources and other embodiments mayuse coherent light sources. By way of non-limiting examples, incoherentlight sources according to some embodiments may include different typesof light emitting diodes (LEDs) such as organic LEDs (OLEDs), quantumdots (QLEDs), nanowire LEDs, and (in)organic semiconductor LEDs. By wayof non-limiting examples, coherent light sources according to someembodiments may include different types of lasers such as semiconductorlasers (e.g., vertical cavity surface emitting lasers (VCSELs), edgeemitting lasers, and distributed-feedback (DFB) laser diodes).Additionally or alternatively, slab-coupled optical waveguide laser(SCOWLs) or other asymmetric single-mode waveguide structures may beused. In some implementations, coherent light sources may compriseorganic lasers, quantum dot lasers, and solid state lasers (e.g., aNd:YAG or ND:Glass laser, pumped by laser diodes or flashlamps). In someembodiments, a laser-diode-pumped fiber laser may be used. A coherentlight source may be passively mode locked to produce ultrashort pulses.There may be more than one type of excitation source for an array ofpixels on an integrated device. In some embodiments, different types ofexcitation sources may be combined. An excitation source may befabricated according to conventional technologies that are used tofabricate a selected type of excitation source.

By way of introduction and without limiting the invention, an examplearrangement of a coherent light source is depicted in FIG. 8-2A. Thedrawing illustrates an analytical instrument 8-200 that may include anultrashort-pulsed laser excitation source 8-210 as the excitationsource. The ultrashort pulsed laser 8-210 may comprise a gain medium8-205 (which may be a solid-state material is some embodiments), a pumpsource for exciting the gain medium (not shown), and at least two cavitymirrors 8-202, 8-204 that define ends of an optical laser cavity. Insome embodiments, there may be one or more additional optical elementsin the laser cavity for purposes of beam shaping, wavelength selection,and/or pulse forming. When operating, the pulsed-laser excitation source8-210 may produce an ultrashort optical pulse 8-220 that circulatesback-and-forth in the laser cavity between the cavity's end mirrors8-202, 8-204 and through the gain medium 8-205. One of the cavitymirrors 8-204 may partially transmit a portion of the circulating pulse,so that a train of optical pulses 8-222 is emitted from the pulsed laser8-210. The emitted pulses may sweep out a beam (indicated by the dashedlines) that is characterized by a beam waist w.

Measured temporal intensity profiles 8-224 of the emitted pulses 8-222may appear as depicted in FIG. 8-2B. In some embodiments, the peakintensity values of the emitted pulses may be approximately equal, andthe profiles 8-224 may have a Gaussian temporal profile, though otherprofiles such as a sech profile may be possible. In some cases, thepulses may not have symmetric temporal profiles and may have othertemporal shapes. In some embodiments, gain and/or loss dynamics mayyield pulses having asymmetric profiles. The duration of each pulse maybe characterized by a full-width-half-maximum (FWHM) value, as indicatedin FIG. 8-2B. Ultrashort optical pulses may have FWHM values less than100 picoseconds.

The pulses emitting from a laser excitation source may be separated byregular intervals T. In some embodiments, T may be determined by activegain and/or loss modulation rates in the laser. For mode-locked lasers,T may be determined by a round-trip travel time between the cavity endmirrors 8-202, 8-204. According to some embodiments, the pulseseparation time T may be between about 1 ns and about 100 ns. In somecases, the pulse separation time T may be between about 0.1 ns and about1 ns. In some implementations, the pulse separation time T may bebetween about 100 ns and about 2 μs.

In some embodiments, an optical system 8-240 may operate on a beam ofpulses 8-222 from a laser excitation source 8-210. For example, theoptical system may include one or more lenses to reshape the beam and/orchange the divergence of the beam. Reshaping of the beam may includeincreasing or decreasing the value of the beam waist and/or changing across-sectional shape of the beam (e.g., elliptical to circular,circular to elliptical, etc.). Changing the divergence of the beam maycomprise converging or diverging the beam flux. In some implementations,the optical system 8-240 may include an attenuator or amplifier tochange the amount of beam energy. In some cases, the optical system mayinclude wavelength filtering elements. In some implementations, theoptical system may include pulse shaping elements, e.g., a pulsestretcher and/or pulse compressor. In some embodiments, the opticalsystem may include one or more nonlinear optical elements, such as asaturable absorber for reducing a pulse length. According to someembodiments, the optical system 8-240 may include one or more elementsthat alter the polarization of pulses from a laser excitation source8-210.

In some implementations, an optical system 8-240 may include a nonlinearcrystal for converting the output wavelength from an excitation source8-210 to a shorter wavelength via frequency doubling or to a longerwavelength via parametric amplification. For example, an output of thelaser may be frequency-doubled in a nonlinear crystal (e.g., inperiodically-poled lithium niobate (PPLN)) or other non-poled nonlinearcrystal. Such a frequency-doubling process may allow more efficientlasers to generate wavelengths more suitable for excitation of selectedfluorophores.

The phrase “characteristic wavelength” or “wavelength” may refer to acentral or predominant wavelength within a limited bandwidth ofradiation produced by an excitation source. In some cases, it may referto a peak wavelength within a bandwidth of radiation produced by anexcitation source. A characteristic wavelength of an excitation sourcemay be selected based upon a choice of luminescent markers or probesthat are used in a bioanalysis device, for example. In someimplementations, the characteristic wavelength of a source of excitationenergy is selected for direct excitation (e.g., single photonexcitation) of a chosen fluorophore. In some implementations, thecharacteristic wavelength of an excitation source is selected forindirect excitation (e.g., multi-photon excitation or harmonicconversion to a wavelength that will provide direct excitation). In someembodiments, excitation radiation may be generated by a light sourcethat is configured to generate excitation energy at a particularwavelength for application to a sample well. In some embodiments, acharacteristic wavelength of the excitation source may be less than acharacteristic wavelength of corresponding emission from the sample. Forexample, an excitation source may emit radiation having a characteristicwavelength between 500 nm and 700 nm (e.g., 515 nm, 532 nm, 563 nm, 594nm, 612 nm, 632 nm, 647 nm). In some embodiments, an excitation sourcemay provide excitation energy centered at two different wavelengths,such as 532 nm and 593 nm for example.

In some embodiments, a pulsed excitation source may be used to excite aluminescent marker in order to measure an emission lifetime of theluminescent marker. This can be useful for distinguishing luminescentmarkers based on emission lifetime rather than emission color orwavelength. As an example, a pulsed excitation source may periodicallyexcite a luminescent marker in order to generate and detect subsequentphoton emission events that are used to determine a lifetime for themarker. Lifetime measurements of luminescent markers may be possiblewhen the excitation pulse from an excitation source transitions from apeak pulse power or intensity to a lower (e.g., nearly extinguished)power or intensity over a duration of time that is less than thelifetime of the luminescent marker. It may be beneficial if theexcitation pulse terminates quickly, so that it does not re-excite theluminescent marker during a post-excitation phase when a lifetime of theluminescent marker is being evaluated. By way of example and notlimitation, the pulse power may drop to approximately 20 dB,approximately 40 dB, approximately 80 dB, or approximately 120 dB lessthan the peak power after 250 picoseconds. In some implementations, thepulse power may drop to approximately 20 dB, approximately 40 dB,approximately 80 dB, or approximately 120 dB less than the peak powerafter 100 picoseconds.

An additional advantage of using ultrashort excitation pulses to exciteluminescent markers is to reduce photo-bleaching of the markers.Applying continuous excitation energy to a marker may bleach and/ordamage a luminescent marker over time. Even though a peak pulse power ofthe excitation source may be considerably higher than a level that wouldrapidly damage a marker at continuous exposure, the use of ultrashortpulses may increase the amount of time and number of useful measurementsbefore the marker becomes damaged by the excitation energy.

When using a pulsed excitation source to discern lifetimes ofluminescent markers, the time between pulses of excitation energy may beas long as or longer than a longest lifetime of the markers in order toobserve and evaluate emission events after each excitation pulse. Forexample, the time interval T (see FIG. 8-2B) between excitation pulsesmay be longer than any emission lifetime of the examined fluorophores.In this case, a subsequent pulse may not arrive before an excitedfluorophore from a previous pulse has had a reasonable amount of time tofluoresce. In some embodiments, the interval T needs to be long enoughto determine a time between an excitation pulse that excites afluorophore and a subsequent photon emitted by the fluorophore aftertermination of excitation pulse and before the next excitation pulse.

Although the interval between excitation pulses T should be long enoughto observe decay properties of the fluorophores, it is also desirablethat T is short enough to allow many measurements to be made in a shortperiod of time. By way of example and not limitation, emission lifetimesof fluorophores used in some applications may be in the range of about100 picoseconds to about 10 nanoseconds. Accordingly, excitation pulsesused to detect and/or discern such lifetimes may have durations (FWHM)ranging from about 25 picoseconds to about 2 nanoseconds, and may beprovided at pulse repetition rates ranging from about 20 MHz to about 1GHz.

In further detail, any suitable techniques for modulating the excitationenergy to create a pulsed excitation source for lifetime measurementsmay be used. Direct modulation of an excitation source, such as a laser,may involve modulating the electrical drive signal of the excitationsource so the emitted power is in the form of pulses. The input powerfor a light source, including the optical pumping power, andexcited-state carrier injection and/or carrier removal from a portion ofthe gain region, may be modulated to affect the gain of the gain medium,allowing the formation of pulses of excitation energy through dynamicgain shaping. Additionally, the quality (Q) factor of the opticalresonator may be modulated by various means to form pulses usingQ-switching techniques. Such Q-switching techniques may be active and/orpassive. The longitudinal modes of a resonant cavity of a laser may bephase-locked to produce a series of pulses of emitted light throughmode-locking. Such mode-locking techniques may be active and/or passive.A laser cavity may include a separate absorbing section to allow formodulation of the carrier density and control of the absorption loss ofthat section, thus providing additional mechanisms for shaping theexcitation pulse. In some embodiments, an optical modulator may be usedto modulate a beam of continuous wave (CW) light to be in the form of apulse of excitation energy. In other embodiments, a signal sent to anacoustic optic modulator (AOM) coupled to an excitation source may beused to change the deflection, intensity, frequency, phase, and/orpolarization of the outputted light to produce a pulsed excitationenergy. AOMs may also be used for continuous wave beam scanning,Q-switching, and/or mode-locking. Although the above techniques aredescribed for creating a pulsed excitation source, any suitable way toproduce a pulsed excitation source may be used for measuring lifetimesof luminescent markers.

In some embodiments, techniques for forming a pulsed excitation sourcesuitable for lifetime measurements may include modulation of an inputelectrical signal that drives photon emission. Some excitation sources(e.g., diode lasers and LEDs) convert an electrical signal, such as aninput current, into a light signal. The characteristics of the lightsignal may depend on characteristics of the electrical signal. Inproducing a pulsed light signal, the electrical signal may vary overtime in order to produce a variable light signal. Modulating theelectrical signal to have a specific waveform may produce an opticalsignal with a specific waveform. The electrical signal may have asinusoidal waveform with a certain frequency and the resulting pulses oflight may occur within time intervals related to the frequency. Forexample, an electrical signal with a 500 MHz frequency may produce alight signal with pulses every 2 nanoseconds. Combined beams produced bydistinct pulsed excitation sources, whether similar or different fromeach other, may have a relative path difference below 1 mm.

In some excitation sources, such as laser diodes, the electrical signalchanges the carrier density and photons are produced through therecombination of electron and hole pairs. The carrier density is relatedto the light signal such that when the carrier density is above athreshold, a substantial number of coherent photons are generated viastimulated emission. Current supplied to a laser diode may injectelectrons or carriers into the device, and thereby increase the carrierdensity. When the carrier density is above a threshold, photons may begenerated at a faster rate than the current supplying the carriers, andthus the carrier density may decrease below the threshold and photongeneration reduces. With the photon generation reduced, the carrierdensity begins to increase again, due to continued current injection andthe absorption of photons, and eventually increases above the thresholdagain. This cycle leads to oscillations of the carrier density aroundthe threshold value for photon generation, resulting in an oscillatinglight signal. These dynamics, known as relaxation oscillations, can leadto artifacts in the light signal due to oscillations of the carrierdensity. When current is initially supplied to a laser, there may beoscillations before the light signal reaches a stable power due tooscillations in the carrier density. When forming a pulsed excitationsource, oscillations of the carrier density may introduce artifacts fora pulsed light signal. For example, the plot in FIG. 8-3 illustrates howthe carrier density may have relaxation oscillations and a correspondinglight signal with oscillating power through modulation by gainswitching. Artifacts from such relaxation oscillations may broaden apulsed light signal and/or produce a tail in the light signal, limitingthe lifetimes that can be detected by such a pulsed light source sincethe excitation signal may overlap with emitted photons by a luminescentmarker.

In some embodiments, techniques for shortening the time duration of anexcitation pulse may be used to reduce the excitation energy required todetect luminescent markers and thereby reduce or delay bleaching andother damage to the luminescent markers. Techniques for shortening thetime duration of the excitation pulse may be used to reduce the powerand/or intensity of the excitation energy after a maximum value or peakof the excitation pulse, allowing the detection of shorter lifetimes.Such techniques may electrically drive the excitation source in order toreduce the excitation power after the peak power. This may suppress atail of the pulse as shown in plot 8-401 of FIG. 8-4. An electricaldriving signal may be tailored to drive the intensity of the pulse ofexcitation energy to zero as quickly as possible after the peak pulse.An example of a tailored electrical driving signal combined with gainswitching is shown in plot 8-402 of FIG. 8-4. Such a technique mayinvolve reversing the sign of an electrical driving signal after thepeak power is produced. Such a tailored electrical driving signal mayproduce an optical output shown in plot 8-403 of FIG. 8-4. Theelectrical signal may be tailored to quickly reduce the carrier densityafter the first relaxation oscillation or first oscillation of theoptical signal. By reducing the carrier density after the firstoscillation, a light pulse of just the first oscillation may begenerated. The electrical signal may be configured to generate a shortpulse that turns the light signal off quickly by reducing the number ofphotons emitted after a peak in the signal, such as shown by the plot inFIG. 8-5 showing the optical output of such an electrical signal. Apicosecond laser diode system may be designed to emit light pulses,according to some embodiments. FIG. 8-6 illustrates a plot of anexemplary light pulse with a peak of 985 mW, a width of 84.3picoseconds, and a signal reduced by approximately 24.3 dB approximately250 picoseconds after the peak. In some embodiments, saturableabsorbers, including semiconductor saturable absorbers (SESAMs) may beused to suppress the optical tail. In such embodiments, using thesaturable absorbers may suppress the optical tail by 3-5 dB or, in someinstances, greater than 5 dB. Reducing the effects of a tail in theexcitation pulse may reduce and/or eliminate any requirements onadditional filtering of the excitation energy, increase the range oflifetimes that can be measured, and/or enable faster pulse rates.Increasing the excitation pulse rate may enable more experiments to beconducted in a given time, which may decrease the time needed to acquireenough statistics to identify a lifetime for a marker labeling a sampleobject.

Additionally, two or more of these techniques may be used together togenerate pulsed excitation energy. For example, pulsed excitation energyemitted from a directly modulated source may be further modified usingoptical modulation techniques. Techniques for modulating the excitationpulse and tailoring the electrical pulse driving signal may be combinedin any suitable way to optimize a pulsed excitation energy forperforming lifetime measurements. A tailored electrical drive signal maybe applied to a pulsed excitation energy from a directly modulatedsource.

In some embodiments, a laser diode having a certain number of wire bondsmay be used as a pulsed excitation source. Laser diodes with more wirebonds may reduce the inductance of the excitation source. Laser diodeshaving a lower inductance (represented by inductor 8-701 of FIG. 8-7)may enable the current into the laser to operate at a higher frequency.As shown in FIG. 8-7, when driven by an 18V pulse in a 50 ohmtransmission line, an Oclaro laser source 8-700 with a 3 ohm seriesresistance (represented by resistor 8-702) and 36 wire bonds has ahigher current at higher frequencies than the laser sources with fewerwire bonds. Selecting a packaging method to minimize inductance mayimprove the power supplied to the excitation source at higherfrequencies, enabling shorter excitation pulses, faster reductions ofoptical power after the peak, and/or increased pulse repetition rate fordetecting luminescent markers.

In some embodiments, a transmission line in combination with anexcitation source may be used for generating light pulses. Thetransmission line may match the impedance of a laser diode in order toimprove performance and/or quality of light pulses. In some embodiments,the transmission line impedance may be 50 ohms. In some instances, theterminating resistance may be similar to the resistance of the line inorder to avoid reflections. Alternatively or additionally, theterminating impedance may be similar to the impedance of the line inorder to avoid reflections. The terminating impedance may less than theimpedance of the line in order to reflect a negative pulse. In otherembodiments, the terminating impedance may have a capacitive orinductive component in order to control the shape of the negativereflection pulse. In other embodiments, the transmission line may allowfor a higher frequency of pulses. FIG. 8-8A illustrates an exampleprototype of a transmission line pulsar, and FIG. 8-8B illustratesexemplary temporal profiles of light pulses obtained with such atransmission line. Using a transmission line may produce electricalpulses having a frequency within a range of 40 MHz to 500 MHz. Atransmission line may be used in combination with a tailored electricalsignal described above in order to produce a pulsed light source withlight pulses having a certain time duration and a specific timeinterval.

Techniques for tailoring the electrical signal to improve the productionof light pulses may include connecting the excitation source to acircuit with a negative bias capability. In some embodiments, a negativebias may be provided on an excitation source after a light pulse emitsto reduce emission of a tail in the light pulse. FIG. 8-9 illustrates anexemplary circuit 8-900 containing a current source 8-901, diode laser8-902, resistor 8-903, capacitor 8-904, and switch 8-905 that may beimplemented to reduce the presence of a tail in a light pulse. Such acircuit 8-900 may create a constant current that bypasses the diodelaser 8-902 when the switch 8-905 is closed, or in a conducting state.When the switch 8-905 is open, the switch 8-905 may have a highresistance and current may flow through the diode laser 8-902. Lightpulses may be generated by opening and closing the switch 8-905 toprovide intermittent current to the diode 8-902 laser. In someinstances, the resistor 8-903 may be sufficiently high and the capacitor8-904 sufficiently small such that there is a voltage across thecapacitor 8-904 when the switch 8-905 is open and the diode laser 8-902emits light. When the switch 8-905 is closed, the voltage across thecapacitor 8-904 will reverse bias the diode laser 8-902. Such a reversebias may reduce or eliminate the presence of a tail in the light pulse.In such instances, the switch 8-905 may be configured to close after thepeak of the light pulse in order to reduce the laser power shortly afterthe peak light pulse. The value of the resistor 8-903 in the circuit8-900 may be selected such that the charge on the capacitor 8-904 willdischarge before the switch is subsequently opened and/or a subsequentlight pulse is generated by the laser diode 8-902.

Additional circuit components may be provided to tailor an electricalsignal of a laser diode in order to produce light pulses. In someembodiments, multiple capacitors, resistors, and voltages may beconnected as a network circuit to control the waveform of an electricalsignal supplied to a laser diode. A controlled waveform may be createdby switching a number of voltages, V1, V2, . . . , VN with correspondingsignals S1, S2, . . . , SN when there are N capacitor sub-circuits. Anexemplary network circuit of four capacitor sub-circuits is shown inFIG. 8-10 where a controlled electrical waveform may be created byswitching voltages V1, V2, V3, and V4 with signals S1, S2, S3, and S4,respectively. In some embodiments, voltages V1, V2, V3, and V4 may bevariable. In the example shown in FIG. 8-10, V4 is negative to the laserand may create a reverse bias depending on signal S4. Timing of thefrequency of light pulses emitted by the laser, the duration of eachlight pulse, and features of each light pulse may be adjusted with thesignal inputs S1, S2, S3, and S4. In some embodiments, additionalresistance may be added to lower the peak current. In such instances,the resistance may be added after one or more of the switches S1, S2,S3, S4. Although FIG. 8-10 shows one configuration with four capacitorsand four voltages, any suitable configuration and any suitable number ofadditional circuit components may be provided to produce a tailoredelectrical signal to the laser diode to generate light pulses forlifetime measurements.

In some embodiments, an electrical signal for generating light pulsesmay use a circuit having discrete components, including radio frequency(RF) and/or microwave components. Discrete components that may beincluded in such a circuit are DC blocks, adaptors, logic gates,terminators, phase shifters, delays, attenuators, combiners, and/or RFamplifiers. Such components may be used to create a positive electricalsignal having a certain amplitude followed by a negative electricalsignal with another amplitude. There may be a delay between the positiveand negative electrical signals. FIG. 8-11A illustrates an exemplarycircuit having one RF amplifier that may be used to produce a tailoredelectrical signal as an output pulse, such as the pulse profile shown inFIG. 8-11B, that may be supplied to an excitation source, such as alaser diode, to emit a light pulse. In other embodiments, a circuit mayproduce multiple electrical signals combined to form an electrical pulsesignal configured to drive an excitation source. Such a circuit mayproduce a differential output which may be used to increase the power ofthe light pulse. By adjusting the discrete components of the circuit,the electrical output signal may be adjusted to produce a light pulsesuitable for lifetime measurements. In an example shown in FIG. 8-12A,two RF amplifiers are used to produce an output pulse signal having aprofile shown in FIG. 8-12B consisting of a positive electrical signalpulse and a corresponding negative electrical signal pulse where thepositive and negative electrical signal pulses overlap and have asimilar width.

In some embodiments, excitation sources may be combined to generatelight pulses for lifetime measurements. Synchronized pulsed sources maybe coupled to a circuit or load over a certain distance. In someembodiments, excitation sources may be coupled in parallel to a circuit.The excitation sources may be from the same source or from multiplesources. In some embodiments with multiple sources, the multiple sourcesmay vary in type of excitation source. When combining sources, it may beimportant to consider the impedance of the circuit and the excitationsources in order to have sufficient power supplied to the excitationsources. Combination of sources may be achieved by using one or more ofthe above-described techniques for producing a pulsed excitation source.FIG. 8-13A illustrates a schematic for combining four different sourceshaving one or more impedance values. FIG. 8-13B shows a plot of current,power efficiency, and voltage as a function of impedance. This exemplaryembodiment shows 4 sources delivering power on 50 ohms transmissionlines and that optimal power delivery occurs when the impedance of theload equals the ratio of the impedance of the individual lines to thenumber of sources.

An excitation source may include a battery or any other power supplyarranged to provide power to the excitation source. For example, anexcitation source may be located in a base instrument and its operatingpower may be received through an integrated bioanalysis device to whichit is coupled (e.g., via conducting power leads). An excitation sourcemay be controlled independently of or in collaboration with control ofan integrated bioanalysis device. As just one example, control signalsfor an excitation source may be provided to the excitation sourcewirelessly or via a wired interconnection (e.g., a USB interconnect)with a personal computer and/or the integrated bioanalysis device.

In some implementations, an excitation source may be operated in atime-gated and/or synchronized manner with one or more sensors of anintegrated device. For example, an excitation source may be turned on toexcite a luminescent marker, and then turned off. The sensor may beturned off while the excitation source is turned on, and then may beturned on for a sampling interval after the excitation source is turnedoff. In some embodiments, the sensor may be turned on for a samplinginterval while the excitation source is turned on.

In some embodiments, an Osram PL 50 mW laser is used, as an example, toprovide an excitation light of 520 nm. Alternatively, an excitationlight of 530 nm or 532 nm is used. Alternatively and/or additionally,Thorlabs LP637-SF70 70 mW laser is used, as an example, to provide anexcitation light of 637 nm. Another example is an Oclaro HL63133 laserdiode that provides excitation at 638 nm. These laser sources or othersuitable lasers sources may be used alone or in combination. One or morelaser sources are used and the excitation light from each source iscombined at a beam combiner and then directed to the assay chip. Eachlight source may be coupled to an optical fiber, the total of which maythen be bundled. The optical characteristics of the light exiting thefiber bundle may be controlled using an optical shaping element (OSE).Optionally, a multiplexer may be used where the laser light frommultiple light sources is multiplexed by polarization, wavelength and/orspatial parameters and sent over a single optical fiber. A diffractiveoptical element (DOE) may be used to spectrally separate the differentwavelengths so that they are directed toward one of the foursub-sensors.

A. Multiple Excitation Sources

Multiple excitation sources may provide light having different energiesor wavelengths to the plurality of sample wells. Each of the multipleexcitation sources may provide light having a different characteristicwavelength or energy. One or more markers may be identified based onwhether light from an excitation source excites a marker such that themarker emits a photon. In this manner, markers may be identified basedon their absorption spectra by measuring the response of a sample afterilluminating the sample with light from the different excitationsources. For example, a sample having a marker may be illuminated withlight from a first excitation source followed by light from a secondexcitation source. If the marker emits luminescence in response to beingilluminated by light from the first excitation source, then the markermay have an absorption spectrum that overlaps with the characteristicwavelength of the first excitation source.

In some embodiments, it is possible to use a plurality of excitationsources for the excitation energy. This plurality of sources may be, forexample, implemented as a diode laser bar comprising multiple diodelaser emitters. In the manufacture of laser diodes, multiple emittersare commonly fabricated lithographically on a single substrate, and thendiced into single-emitter pieces for individual packaging. But it isalso possible to dice the substrate into pieces with a plurality ofemitters. In some embodiments, the emitters are nearly identical, andmay be spaced equally from each other to lithographic tolerances,typically of the order of 0.1 micrometer.

VI. METHOD OF USE, INSTRUMENT OPERATION AND USER INTERFACE

The instrument 2-120 may be controlled using software and/or hardware.For example, the instrument may be controlled using a processing device1-123, such as an ASIC, an FPGA and/or a general purpose processorexecuting software.

FIG. 9-1 illustrates a flowchart of operation of the instrument 2-120according to some embodiments. After a user has acquired a specimen toanalyze, the user begins a new analysis at act 9-101. This may be doneby providing an indication to the instrument 2-120 via the userinterface 2-125 by, e.g., pressing a button. At act 9-103, theinstrument 2-120 checks whether the assay chip 2-110 from a previouslyperformed analysis is still inserted in the instrument 2-120. If it isdetermined that an old assay chip is present, then the power toexcitation source may be turned off at act 9-105, the user is promptedat act 9-107 to eject the previous assay chip using an indicator of theuser interface 2-125 and the instrument 2-120 waits for the old assaychip to be ejected at act 9-109.

When the previous assay chip is ejected by the user, or if theinstrument 2-120 determined at act 9-103 that the previous assay chipwas already removed, the user is prompted to insert a new assay chip2-110 for the new analysis at act 9-111. The instrument 2-120 then waitsfor the new assay chip 2-110 to be inserted at act 9-113. When the userinserts the new assay chip, the user is prompted at act 9-115 by anindicator of the user interface 2-125 to place the specimen to beanalyzed onto the exposed top surface of the assay chip 2-110 and alsoprompted to close the lid on the instrument 2-120. The instrument 2-120then waits for the lid to be closed at act 9-117. When the lid is closedby the user, at act 9-119 the excitation source may be driven to produceexcitation energy for exciting the sample portions of the specimenpresent in the sample wells of the assay chip 2-110. At act 9-121, theemission energy from the samples is detected by the sensor 2-122 anddata from the sensor 2-122 is streamed to the processing device 2-123for analysis. In some embodiments, the data may be streamed to externalcomputing device 2-130. At act 2-123, the instrument 2-120 checkswhether the data acquisition is complete. The data acquisition may becomplete after a particular length of time, a particular number ofexcitation pulses from the excitation source or one a particular targethas been identified. When the data acquisition is completed, the dataanalysis is finished at 9-125.

FIG. 9-2 illustrates an example self-calibration routine according tosome embodiments. The calibration routine may be executed at anysuitable time prior to the analysis of a specimen. For example, it maybe done once by the manufacturer for each instrument prior to shipmentto the end user. Alternatively, the end user may perform a calibrationat any suitable time. As discussed above, the instrument 2-120 iscapable of distinguishing between emission energy having differentwavelengths emitted from different samples. The instrument 2-120 and/orcomputing device 2-130 may be calibrated with calibration associatedwith each particular color of light associated with, for example, aluminescent tag used to tag molecules of a specimen being analyzed. Inthis way, the precise output signal associated with a particular colormay be determined.

To calibrate the device, a calibration specimen associated with a singleluminescent tag is provided to the instrument 2-120 one at a time. Theself-calibration begins at act 9-201 when a user places a specimencomprising luminescent tags that emit emission energy of a singlewavelength on an assay chip 2-110 and inserts the assay chip 2-110 intothe instrument 2-120. Using the user interface 2-125, the user instructsthe instrument 2-120 to begin the self-calibration. In response, at act9-203, the instrument 2-120 runs the calibration analysis byilluminating the assay chip 2-110 with excitation energy and measuringthe single wavelength emission energy from the calibration specimen. Theinstrument 2-120 may then, at act 9-205, save the detection patternmeasured on the array of sub-sensors of the sensor 2-122 for each pixelof the sensor array. The detection pattern for each luminescent tag maybe considered a detection signature associated with the luminescent tag.In this way, the signatures may be used as a training data set used toanalyze the data received from unknown samples analyzed in subsequentanalysis runs.

The above calibration routine may then be executed for every calibrationspecimen associated with a single luminescent tag. In this way, eachsensor 2-122 of the array of pixels is associated with calibration datathat may be used to determine the luminescent tag present in a samplewell during a subsequent analysis implemented at act 9-207 after thecompetition of the calibration routine.

FIG. 9-3 further illustrates how the calibration data may be acquiredand used to analyze the data according to some embodiments. At act 9-301calibration data is obtained from the sensors. This may be done usingthe aforementioned self-calibration routine. At act 9-303, atransformation matrix is generated based on the calibration data. Thetransformation matrix maps sensor data to the emission wavelength of asample and is a m×n matrix, where m is the number of luminescent tagswith different emission wavelengths and n is the number of sub-sensorsused to detect the emission energy per pixel. Thus, each column of thetransformation matrix represents the calibration values for the sensor.For example, if there are four sub-sensors per pixel and five differentluminescent tags, then the transformation matrix is a 4×5 matrix (i.e.,four rows and five columns) and each column is associated with adifferent luminescent tag, the values in the column corresponding to themeasured values obtained from the sub-sensors during theself-calibration routine. In some embodiments, each pixel may have itsown transformation matrix. In other embodiments, the calibration datafrom at least some of the pixels may be averaged and all the pixels maythen use the same transformation matrix based on the averaged data.

At act 9-305, the analysis data associated with a bioassay is obtainedfrom the sensors. This may be done in any of the ways described above.At act 9-307, the wavelength of the emission energy and/or the identityof the luminescent tag may be determined using the transformation matrixand the analysis data. This may be done in any suitable way. In someembodiments, the analysis data is multiplied by the pseudo-inverse ofthe transformation matrix, resulting in a m×1 vector. The luminescenttag associated with the vector component with the maximum value may thenbe identified as the luminescent tag present in the sample well.Embodiments are not limited to this technique. In some embodiments, toprevent possible pathologies that may arise when the inverse of a matrixwith small values is taken, a constrained optimization routine, such asa least square method or a maximum likelihood technique, may beperformed to determine the luminescent tag present in the sample well.

The foregoing method of using the calibration data to analyze data fromthe sensors may be implement by any suitable processor. For example,processing device 2-123 of the instrument 2-120 may perform theanalysis, or computing device 2-130 may perform the analysis.

VII. EXAMPLE MEASUREMENTS WITH THE ASSAY CHIP AND INSTRUMENT

Measurements for detecting, analyzing, and/or probing molecules in asample may be obtained using any combination of the assay chip andinstrument described in the present application. The excitation sourcemay be a pulsed excitation source or, in some instances, a continuouswave source. A luminescent marker tagged to a specific sample mayindicate the presence of the sample. Luminescent markers may bedistinguished by excitation energy, emission energy, and/or the lifetimeof emission energy emitted by a marker. Markers with similarluminescence emission wavelengths may be identified by determining thelifetime for each marker. Additionally, markers with similar lifetimesmay be identified by the luminescence emission wavelength for eachmarker. By using markers, where markers are identified by a combinationof the temporal and/or spectral properties of the emitted luminescence,a quantitative analysis and/or identification of markers and associatedsamples can be performed.

Lifetime measurements may be used to determine that a marker is presentin a sample well. The lifetime of a luminescent marker may be identifiedby performing multiple experiments where the luminescent marker isexcited into an excited state and then the time when a photon emits ismeasured. The excitation source is pulsed to produce a pulse ofexcitation energy and directed at the marker. The time between theexcitation pulse and a subsequent photon emission event from aluminescent marker is measured. By repeating such experiments with aplurality of excitation pulses, the number of instances a photon emitswithin a particular time interval may be determined. Such results maypopulate a histogram representing the number of photon emission eventsthat occur within a series of discrete time intervals or time bins. Thenumber of time bins and/or the time interval of each bin may be adjustedto identify a particular set of lifetimes and/or markers.

What follows is a description of example measurements that may be madeto identify luminescent markers in some embodiments. Specifically,examples of distinguishing luminescent markers using only a luminescentlifetime measurement, a joint spectral and luminescent lifetimemeasurement, and only a luminescent lifetime measurement, but using twodifferent excitation energies are discussed. Embodiments are not limitedto the examples detailed below. For example, some embodiments mayidentify the luminescent markers using only spectral measurements.

Any suitable luminescent markers may be used. In some embodiments,commercially available fluorophores may be used. By way of example andnot limitation, the following fluorophores may be used: Atto Rho14(“ATRho14”), Dylight 650 (“D650”), SetaTau 647 (“ST647”), CF 633(“C633”), CF 647 (“C647”), Alexa fluor 647 (“AF647”), BODIPY 630/650(“B630”), CF 640R (“C640R”) and/or Atto 647N (“AT647N”).

Additionally and/or optionally, luminescent markers may be modified inany suitable way to increase the speed and accuracy of the sampleanalysis process. For example, a photostabilizer may be conjugated to aluminescent marker. Examples of photostabilizers include but are notlimited to oxygen scavengers or triplet-state quenchers. Conjugatingphotostabilizers to the luminescent marker may increase the rate ofphotons emitted and may also reduce a “blinking” effect where theluminescent marker does not emit photons. In some embodiments, when abiological event occurs on the millisecond scale, an increased rate ofphoton emission may increase the probability of detection of thebiological event. Increased rates of photon events may subsequentlyincrease the signal-to-noise ratio of luminescence signal and increasethe rate at which lifetime measurements are made, leading to a fasterand more accurate sample analysis.

Furthermore, the environment in a sample well of an integrated devicemay be tuned to engineer the lifetime of the markers as needed. This canbe achieved by recognizing that the lifetime of a marker is effected bythe density of state of the marker, which can be tuned using theenvironment. For example, the farther a marker is from the bottom metallayer of the sample well, the longer the lifetime. Accordingly, toincrease the lifetime of a marker, the depth of a bottom surface of asample well, such as a divot, may extend a certain distance from a metallayer. Also, the materials used to form the sample well can affect thelifetime of the markers. While different markers typically have theirlifetimes shifted in the same direction (e.g., either longer orshorter), the affect may scale differently for different markers.Accordingly, two markers that cannot be distinguished via lifetimemeasurements in free-space may be engineered to be distinguishable byfabricating the sample well environment to adjust the lifetimes of thevarious markers.

A. Single Molecule Detection and Sequencing

According to an aspect of the present application, a single molecule canbe identified (e.g., distinguished from other possible molecules in areaction sample) based on one or more properties of a series of photonsthat are emitted from the molecule when it is exposed to a plurality ofseparate light pulses. In some embodiments, the molecule is labelledwith a luminescent marker. In some embodiments, the luminescent markeris a fluorophore. In some embodiments, the luminescent marker can beidentified or distinguished based on a property of the luminescentmarker. Properties of a luminescent label (e.g., a fluorophore) include,but are not limited to luminescent lifetimes, absorption spectra,emission spectra, luminescence quantum yield, and luminescent intensity,and combinations of two or more thereof.

A biological sample may be processed in preparation for detection (e.g.,sequencing). Such processing can include isolation and/or purificationof the biomolecule (e.g., nucleic acid molecule) from the biologicalsample, and generation of more copies of the biomolecule. In someexamples, one or more nucleic acid molecules are isolated and purifiedform a bodily fluid or tissue of the subject, and amplified throughnucleic acid amplification, such as polymerase chain reaction (PCR).Then, the one or more nucleic acids molecules or subunits thereof can beidentified, such as through sequencing. However, in some embodimentsnucleic acid samples can be evaluated (e.g., sequenced) as described inthis application without requiring amplification.

Sequencing can include the determination of individual subunits of atemplate biomolecule (e.g., nucleic acid molecule) by synthesizinganother biomolecule that is complementary or analogous to the template,such as by synthesizing a nucleic acid molecule that is complementary toa template nucleic acid molecule and identifying the incorporation ofnucleotides with time (e.g., sequencing by synthesis). As analternative, sequencing can include the direct identification ofindividual subunits of the biomolecule.

During sequencing, a polymerizing enzyme may couple (e.g., attach) to apriming location of a target nucleic acid molecule. The priming locationcan be a primer that is complementary to the target nucleic acidmolecule. As an alternative the priming location is a gap or nick thatis provided within a double stranded segment of the target nucleic acidmolecule. A gap or nick can be from 0 to at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 20, 30, or 40 nucleotides in length. A nick can provide abreak in one strand of a double stranded sequence, which can provide apriming location for a polymerizing enzyme, such as, for example, astrand displacing polymerase enzyme.

In some cases, a sequencing primer can be annealed to a target nucleicacid molecule that may or may not be immobilized to a solid support,such as a sample well. In some embodiments, a sequencing primer may beimmobilized to a solid support and hybridization of the target nucleicacid molecule also immobilizes the target nucleic acid molecule to thesolid support. Via the action of an enzyme (e.g., a polymerase) capableof adding or incorporating a nucleotide to the primer, nucleotides canbe added to the primer in 5′ to 3′, template bound fashion. Suchincorporation of nucleotides to a primer (e.g., via the action of apolymerase) can generally be referred to as a primer extension reaction.Each nucleotide can be associated with a detectable tag that can bedetected and used to determine each nucleotide incorporated into theprimer and, thus, a sequence of the newly synthesized nucleic acidmolecule. Via sequence complementarity of the newly synthesized nucleicacid molecule, the sequence of the target nucleic acid molecule can alsobe determined. In some cases, annealing of a sequencing primer to atarget nucleic acid molecule and incorporation of nucleotides to thesequencing primer can occur at similar reaction conditions (e.g., thesame or similar reaction temperature) or at differing reactionconditions (e.g., different reaction temperatures). Moreover, somesequencing by synthesis methods can include the presence of a populationof target nucleic acid molecules (e.g, copies of a target nucleic acid)and/or a step of amplification of the target nucleic acid to achieve apopulation of target nucleic acids.

Embodiments are capable of sequencing single nucleic acid molecules withhigh accuracy and long read length. In some embodiments, the targetnucleic acid molecule used in single molecule sequencing is asingle-stranded target nucleic acid (e.g. deoxyribonucleic acid (DNA),DNA derivatives, ribonucleic acid (RNA), RNA derivatives) template thatis added or immobilized to a sample well containing at least oneadditional component of a sequencing reaction (e.g., a polymerase suchas, a DNA polymerase, a sequencing primer) immobilized or attached to asolid support such as the bottom of the sample well. The target nucleicacid molecule or the polymerase can be attached to a sample wall, suchas at the bottom of the sample well directly or through a linker. Thesample well can also contain any other reagents needed for nucleic acidsynthesis via a primer extension reaction, such as, for example suitablebuffers, co-factors, enzymes (e.g., a polymerase) anddeoxyribonucleoside polyphosphates, such as, e.g., deoxyribonucleosidetriphosphates, including deoxyadenosine triphosphate (dATP),deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP),deoxyuridine triphosphate (dUTP) and deoxythymidine triphosphate (dTTP)dNTPs, that include luminescent tags, such as fluorophores. Each classof dNTPs (e.g. adenine-containing dNTPs (e.g., dATP),cytosine-containing dNTPs (e.g., dCTP), guanine-containing dNTPs (e.g.,dGTP), uracil-containing dNTPs (e.g., dUTPs) and thymine-containingdNTPs (e.g., dTTP)) is conjugated to a distinct luminescent tag suchthat detection of light emitted from the tag indicates the identity ofthe dNTP that was incorporated into the newly synthesized nucleic acid.Emitted light from the luminescent tag can be detected and attributed toits appropriate luminescent tag (and, thus, associated dNTP) via anysuitable device and/or method, including such devices and methods fordetection described elsewhere herein. The luminescent tag may beconjugated to the dNTP at any position such that the presence of theluminescent tag does not inhibit the incorporation of the dNTP into thenewly synthesized nucleic acid strand or the activity of the polymerase.In some embodiments, the luminescent tag is conjugated to the terminalphosphate (the gamma phosphate) of the dNTP.

The single-stranded target nucleic acid template can be contacted with asequencing primer, dNTPs, polymerase and other reagents necessary fornucleic acid synthesis. In some embodiments, all appropriate dNTPs canbe contacted with the single-stranded target nucleic acid templatesimultaneously (e.g., all dNTPs are simultaneously present) such thatincorporation of dNTPs can occur continuously. In other embodiments, thedNTPs can be contacted with the single-stranded target nucleic acidtemplate sequentially, where the single-stranded target nucleic acidtemplate is contacted with each appropriate dNTP separately, withwashing steps in between contact of the single-stranded target nucleicacid template with differing dNTPs. Such a cycle of contacting thesingle-stranded target nucleic acid template with each dNTP separatelyfollowed by washing can be repeated for each successive base position ofthe single-stranded target nucleic acid template to be identified.

The sequencing primer anneals to the single-stranded target nucleic acidtemplate and the polymerase consecutively incorporates the dNTPs (orother deoxyribonucleoside polyphosphate) to the primer via thesingle-stranded target nucleic acid template. The unique luminescent tagassociated with each incorporated dNTP can be excited with theappropriate excitation light during or after incorporation of the dNTPto the primer and its emission can be subsequently detected, using, anysuitable device(s) and/or method(s), including devices and methods fordetection described elsewhere herein. Detection of a particular emissionof light can be attributed to a particular dNTP incorporated. Thesequence obtained from the collection of detected luminescent tags canthen be used to determine the sequence of the single-stranded targetnucleic acid template via sequence complementarity.

While the present disclosure makes reference to dNTPs, devices, systemsand methods provided herein may be used with various types ofnucleotides, such as ribonucleotides and deoxyribonucleotides (e.g.,deoxyribonucleoside polyphophates with at least 4, 5, 6, 7, 8, 9, or 10phosphate groups). Such ribonucleotides and deoxyribonucleotides caninclude various types of tags (or markers) and linkers.

Signals emitted upon the incorporation of nucleosides can be stored inmemory and processed at a later point in time to determine the sequenceof the target nucleic acid template. This may include comparing thesignals to a reference signals to determine the identities of theincorporated nucleosides as a function of time. Alternative or inaddition to, signal emitted upon the incorporation of nucleoside can becollected and processed in real time (i.e., upon nucleosideincorporation) to determine the sequence of the target nucleic acidtemplate in real time.

Nucleic acid sequencing of a plurality of single-stranded target nucleicacid templates may be completed where multiple sample wells areavailable, as is the case in devices described elsewhere herein. Eachsample well can be provided with a single-stranded target nucleic acidtemplate and a sequencing reaction can be completed in each sample well.Each of the sample wells may be contacted with the appropriate reagents(e.g., dNTPs, sequencing primers, polymerase, co-factors, appropriatebuffers, etc.) necessary for nucleic acid synthesis during a primerextension reaction and the sequencing reaction can proceed in eachsample well. In some embodiments, the multiple sample wells arecontacted with all appropriate dNTPs simultaneously. In otherembodiments, the multiple sample wells are contacted with eachappropriate dNTP separately and each washed in between contact withdifferent dNTPs. Incorporated dNTPs can be detected in each sample welland a sequence determined for the single-stranded target nucleic acid ineach sample well as is described above.

Embodiments directed toward single molecule RNA sequencing may use anyreverse transcriptase that is capable of synthesizing complementary DNA(cDNA) from an RNA template. In such embodiments, a reversetranscriptase can function in a manner similar to polymerase in thatcDNA can be synthesized from an RNA template via the incorporation ofdNTPs to a reverse transcription primer annealed to an RNA template. ThecDNA can then participate in a sequencing reaction and its sequencedetermined as described above. The determined sequence of the cDNA canthen be used, via sequence complementarity, to determine the sequence ofthe original RNA template. Examples of reverse transcriptases includeMoloney Murine Leukemia Virus reverse transcriptase (M-MLV), avianmyeloblastosis virus (AMV) reverse transcriptase, human immunodeficiencyvirus reverse transcriptase (HIV-1) and telomerase reversetranscriptase.

Sequence reads can be used to reconstruct a longer region of a genome ofa subject (e.g., by alignment). Reads can be used to reconstructchromosomal regions, whole chromosomes, or the whole genome. Sequencereads or a larger sequence generated from such reads can be used toanalyze a genome of a subject, such as to identify variants orpolymorphisms. Examples of variants include, but are not limited to,single nucleotide polymorphisms (SNPs) including tandem SNPs,small-scale multi-base deletions or insertions, also referred to asindels or deletion insertion polymorphisms (DIPs), Multi-NucleotidePolymorphisms (MNPs), Short Tandem Repeats (STRs), deletions, includingmicrodeletions, insertions, including microinsertions, structuralvariations, including duplications, inversions, translocations,multiplications, complex multi-site variants, copy number variations(CNV). Genomic sequences can comprise combinations of variants. Forexample, genomic sequences can encompass the combination of one or moreSNPs and one or more CNVs.

In some embodiments, the molecules are identified or distinguished basedon luminescent lifetime. In some embodiments, the molecules areidentified or distinguished based on luminescent intensity. In someembodiments, the molecules are identified or distinguished based on thewavelength of the delivered excitation energy necessary to observe anemitted photon. In some embodiments, the molecules are identified ordistinguished based on the wavelength of an emitted photon. In someembodiments, the molecules are identified or distinguished based on bothluminescent lifetime and the wavelength of the delivered excitationenergy necessary to observe an emitted photon. In some embodiments, themolecules are identified or distinguished based on both a luminescentintensity and the wavelength of the delivered excitation energynecessary to observe an emitted photon. In some embodiments, themolecules are identified or distinguished based on luminescent lifetime,luminescent intensity, and the wavelength of the delivered excitationenergy necessary to observe an emitted photon. In some embodiments, themolecules are identified or distinguished based on both luminescentlifetime and the wavelength of an emitted photon. In some embodiments,the molecules are identified or distinguished based on both aluminescent intensity and the wavelength of an emitted photon. In someembodiments, the molecules are identified or distinguished based onluminescent lifetime, luminescent intensity, and the wavelength of anemitted photon.

In certain embodiments, different types of molecules in a reactionmixture or experiment are labeled with different luminescent markers. Insome embodiments, the different markers have different luminescentproperties which can be distinguished. In some embodiments, thedifferent markers are distinguished by having different luminescentlifetimes, different luminescent intensities, different wavelengths ofemitted photons, or a combination thereof. The presence of a pluralityof types of molecules with different luminescent markers may allow fordifferent steps of a complex reaction to be monitored, or for differentcomponents of a complex reaction product to be identified. In someembodiments, the order in which the different types of molecules reactor interact can be determined.

In certain embodiments, the luminescent properties of a plurality oftypes of molecules with different luminescent markers are used toidentify the sequence of a biomolecule, such as a nucleic acid orprotein. In some embodiments, the luminescent properties of a pluralityof types of molecules with different luminescent markers are used toidentify single molecules as they are incorporated during the synthesisof a biomolecule. In some embodiments, the luminescent properties of aplurality of types of nucleotides with different luminescent markers areused to identify single nucleotides as they are incorporated during asequencing reaction. In some embodiments, methods, compositions, anddevices described in the application can be used to identify a series ofnucleotides that are incorporated into a template-dependent nucleic acidsequencing reaction product synthesized by a polymerase enzyme.

In certain embodiments, the template-dependent nucleic acid sequencingproduct is carried out by naturally occurring nucleic acid polymerases.In some embodiments, the polymerase is a mutant or modified variant of anaturally occurring polymerase. In some embodiments, thetemplate-dependent nucleic acid sequence product will comprise one ormore nucleotide segments complementary to the template nucleic acidstrand. In one aspect, the application provides a method of determiningthe sequence of a template (or target) nucleic acid strand bydetermining the sequence of its complementary nucleic acid strand.

In another aspect, the application provides methods of sequencing targetnucleic acids by sequencing a plurality of nucleic acid fragments,wherein the target nucleic acid comprises the fragments. In certainembodiments, the method comprises combining a plurality of fragmentsequences to provide a sequence or partial sequence for the parenttarget nucleic acid. In some embodiments, the step of combining isperformed by computer hardware and software. The methods describedherein may allow for a set of related target nucleic acids, such as anentire chromosome or genome to be sequenced.

The term “genome” generally refers to an entirety of an organism'shereditary information. A genome can be encoded either in DNA or in RNA.A genome can comprise coding regions that code for proteins as well asnon-coding regions. A genome can include the sequence of all chromosomestogether in an organism. For example, the human genome has a total of 46chromosomes. The sequence of all of these together constitutes the humangenome. In some embodiments, the sequence of an entire genome isdetermined. However, in some embodiments, sequence information for asubset of a genome (e.g., one or a few chromosomes, or regions thereof)or for one or a few genes (or fragments thereof) are sufficient fordiagnostic, prognostic, and/or therapeutic applications.

In some cases, a sequencing primer can be annealed to a target nucleicacid molecule that may or may not be immobilized to a solid support,such as a sample well (e.g., nanoaperture). In some embodiments, asequencing primer may be immobilized to a solid support andhybridization of the target nucleic acid molecule also immobilizes thetarget nucleic acid molecule to the solid support. In some embodiments,a polymerase is immobilized to a solid support and soluble primer andtarget nucleic acid are contacted to the polymerase. However, in someembodiments a complex comprising a polymerase, a target nucleic acid anda primer is formed in solution and the complex is immobilized to a solidsupport (e.g., via immobilization of the polymerase, primer, and/ortarget nucleic acid).

Under appropriate conditions, a polymerase enzyme that is contacted toan annealed primer/target nucleic acid can add or incorporate one ormore nucleotides onto the primer, and nucleotides can be added to theprimer in a 5′ to 3′, template bound fashion. Such incorporation ofnucleotides onto a primer (e.g., via the action of a polymerase) cangenerally be referred to as a primer extension reaction. Each nucleotidecan be associated with a detectable tag that can be detected andidentified (e.g., based on its luminescent lifetime, emission spectra,absorption spectra, and/or other characteristics) and used to determineeach nucleotide incorporated into the primer and, thus, a sequence ofthe newly synthesized nucleic acid molecule. Via sequencecomplementarity of the newly synthesized nucleic acid molecule, thesequence of the target nucleic acid molecule can also be determined. Insome embodiments, sequencing by synthesis methods can include thepresence of a population of target nucleic acid molecules (e.g., copiesof a target nucleic acid) and/or a step of amplification of the targetnucleic acid to achieve a population of target nucleic acids. However,in some embodiments sequencing by synthesis is used to determine thesequence of a single molecule in each reaction that is being evaluated(and nucleic acid amplification is not required to prepare the targettemplate for sequencing). In some embodiments, a plurality of singlemolecule sequencing reactions are performed in parallel (e.g., on asingle integrated device or chip) according to aspects of the presentapplication.

Embodiments are capable of sequencing single nucleic acid molecules withhigh accuracy and long read lengths, such as an accuracy of at leastabout 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%,99.99%, 99.999%, or 99.9999%, and/or read lengths greater than or equalto about 10 base pairs (bp), 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500bp, 1000 bp, 10,000 bp, 20,000 bp, 30,000 bp, 40,000 bp, 50,000 bp, or100,000 bp.

The target nucleic acid molecule or the polymerase can be attached to asample wall, such as at the bottom of the sample well directly orthrough a linker. The sample well (e.g., nanoaperture) also can containany other reagents needed for nucleic acid synthesis via a primerextension reaction, such as, for example suitable buffers, co-factors,enzymes (e.g., a polymerase) and deoxyribonucleoside polyphosphates,such as, e.g., deoxyribonucleoside triphosphates, includingdeoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP),deoxyguanosine triphosphate (dGTP), deoxyuridine triphosphate (dUTP) anddeoxythymidine triphosphate (dTTP) dNTPs, that include luminescent tags,such as fluorophores. In some embodiments, each class of dNTPs (e.g.adenine-containing dNTPs (e.g., dATP), cytosine-containing dNTPs (e.g.,dCTP), guanine-containing dNTPs (e.g., dGTP), uracil-containing dNTPs(e.g., dUTPs) and thymine-containing dNTPs (e.g., dTTP)) is conjugatedto a distinct luminescent tag such that detection of light emitted fromthe tag indicates the identity of the dNTP that was incorporated intothe newly synthesized nucleic acid. Emitted light from the luminescenttag can be detected and attributed to its appropriate luminescent tag(and, thus, associated dNTP) via any suitable device and/or method,including such devices and methods for detection described elsewhereherein. The luminescent tag may be conjugated to the dNTP at anyposition such that the presence of the luminescent tag does not inhibitthe incorporation of the dNTP into the newly synthesized nucleic acidstrand or the activity of the polymerase. In some embodiments, theluminescent tag is conjugated to the terminal phosphate (the gammaphosphate) of the dNTP.

In some embodiments, the sequencing primer anneals to thesingle-stranded target nucleic acid template and the polymeraseconsecutively incorporates the dNTPs (or other deoxyribonucleosidepolyphosphate) to the primer via the single-stranded target nucleic acidtemplate. The unique luminescent tag associated with each incorporateddNTP can be excited with the appropriate excitation light during orafter incorporation of the dNTP to the primer and its emission can besubsequently detected, using, any suitable device(s) and/or method(s),including devices and methods for detection described elsewhere herein.Detection of a particular emission of light (e.g., having a particularemission lifetime, intensity, and/or combination thereof) can beattributed to a particular dNTP incorporated. The sequence obtained fromthe collection of detected luminescent tags can then be used todetermine the sequence of the single-stranded target nucleic acidtemplate via sequence complementarity.

While the present disclosure makes reference to dNTPs, devices, systemsand methods provided herein may be used with various types ofnucleotides, such as ribonucleotides and deoxyribonucleotides (e.g.,deoxyribonucleoside polyphophates with at least 4, 5, 6, 7, 8, 9, or 10phosphate groups). Such ribonucleotides and deoxyribonucleotides caninclude various types of tags (or markers) and linkers.

As an example, FIG. 10-1 schematically illustrates the setup of a singlemolecule nucleic acid sequencing method. The example is not meant tolimit the invention in any way. 610 is a sample well (e.g.,nanoaperture) configured to contain a single complex comprising anucleic acid polymerase 601, a target nucleic acid 602 to be sequenced,and a primer 604. In this example, a bottom region of sample well 610 isdepicted as a target volume 620. In FIG. 10-1 the complex comprisingpolymerase 601 is confined in target volume 620. The complex mayoptionally be immobilized by attachment to a surface of the sample well.In this example the complex is immobilized by a linker 603 comprisingone or more biomolecules (e.g., biotin) suitable for attaching thelinker to the polymerase 601.

The volume of the sample well also contains a reaction mixture withsuitable solvent, buffers, and other additives necessary for thepolymerase complex to synthesize a nucleic acid strand. The reactionmixture also contains a plurality of types of luminescently labelednucleotides. Each type of nucleotide is represented by the symbols *-A,@-T, $-G, #-C, wherein A, T, G, and C represent the nucleotide base, andthe symbols *, @, $, and # represent a unique luminescent label attachedto each nucleotide, through linker -. In FIG. 10-1, a #-C nucleotide iscurrently being incorporated into the complementary strand 602. Theincorporated nucleotide is located within the target volume 620.

FIG. 10-1 also indicates with arrows the concept of an excitation energybeing delivered to a vicinity of the target volume, and luminescencebeing emitted towards a detector. The arrows are schematic, and are notmeant to indicate the particular orientation of excitation energydelivery or luminescence. Some luminescences may emit on a vector whichis not directed to the detector (e.g., towards the sidewall of thesample well) and may not be detected.

FIG. 10-2 schematically illustrates a sequencing process in a singlesample well over time. Stages A through D depict a sample well with apolymerase complex as in FIG. 10-1. Stage A depicts the initial statebefore any nucleotides have been added to the primer. Stage B depictsthe incorporation event of a luminescently labeled nucleotide (#-C).Stage C, depicts the period between incorporation events. In thisexample, nucleotide C has been added to the primer, and the label andlinker previously attached to the luminescently labeled nucleotide (#-C)has been cleaved. Stage D depicts a second incorporation event of aluminescently labeled nucleotide (*-A). The complementary strand afterStage D consists of the primer, a C nucleotide, and an A nucleotide.

Stage A and C, both depict the periods before or between incorporationevents, which are indicated in this example to last for about 10milliseconds. In stages A and C, because there is no nucleotide beingincorporated, there is no luminescently labeled nucleotide in the targetvolume (not drawn in FIG. 10-2), though background luminescence orspurious luminescence from a luminescently labeled nucleotide which isnot being incorporated may be detected. Stage B and D show incorporationevents of different nucleotides (#-C, and *-A, respectively). In thisexample these events are also indicated to last for about 10milliseconds.

The row labeled “Raw bin data” depicts the data generated during eachStage. Throughout the example experiment, a plurality of pulses of lightis delivered to the vicinity of the target volume. For each pulse adetector is configured to record any emitted photon received by thedetector, and assign the detected photon to a time bin based on the timeduration since the last pulse of excitation energy. In this examplethere are 3 bins, and the “Raw bin data” records a value of 1 (shortestbars), 2 (medium bars), or 3 (longest bars), corresponding to theshortest, middle, and longest bins, respectively. Each bar indicatesdetection of an emitted photon.

Since there is no luminescently labeled nucleotide present in the targetvolume for Stage A or C, there are no photons detected. For each ofStage B and D a plurality of luminescences is detected during theincorporation event. Luminescent label # has a shorter luminescencelifetime than luminescent label *. The Stage B data is thus depicted ashaving recorded lower average bin values, than Stage D where the binvalues are higher.

The row labeled “Processed data” depicts raw data which has beenprocessed to indicate the number (counts) of emitted photons at timesrelative to each pulse. In this example the data is only processed todetermine luminescent lifetime, but the data may also be evaluated forother luminescent properties, such as luminescent intensity or thewavelength of the absorbed or emitted photons. The exemplary processeddata approximates an exponential decay curve characteristic for theluminescence lifetime of the luminescent marker in the target volume.Because luminescent label # has a shorter luminescence lifetime thanluminescent label *, the processed data for Stage B has fewer counts atlonger time durations, while the processed data for Stage D hasrelatively more counts at longer time durations.

The example experiment of FIG. 10-2 would identify the first twonucleotides added to the complementary strand as CA. For DNA, thesequence of the target strand immediately after the region annealed tothe primer would thus be identified as GT. In this example thenucleotides C and A could be distinguished from amongst the plurality ofC, G, T, and A, based on luminescent lifetime alone. In someembodiments, other properties, such as the luminescent intensity or thewavelength of the absorbed or emitted photons may be necessary todistinguish one or more particular nucleotides.

B. Luminescent Properties

As described herein, a luminescent molecule is a molecule that absorbsone or more photons and may subsequently emit one or more photons afterone or more time durations. The luminescence of the molecule isdescribed by several parameters, including but not limited toluminescent lifetime, absorption and/or emission spectra, luminescentquantum yield, and luminescent intensity.

The emitted photon from a luminescent emission event will emit at awavelength within a spectral range of possible wavelengths. Typicallythe emitted photon has a longer wavelength (e.g., has less energy or isred-shifted) compared to the wavelength of the excitation photon. Incertain embodiments, a molecule is identified my measuring thewavelength of an emitted photon. In certain embodiments, a molecule isidentified by measuring the wavelength of a plurality of emittedphotons. In certain embodiments, a molecule is identified by measuringthe emission spectrum.

Luminescent lifetime refers to a time duration (e.g., emission decaytime) associated with an excitation event and an emission event. In someembodiments, luminescent lifetime is expressed as the constant in anequation of exponential decay. In some embodiments, wherein there areone or more pulse events delivering excitation energy, the time durationis the time between the pulse and the subsequent emission event.

Luminescent quantum yield refers to the fraction of excitation events ata given wavelength or within a given spectral range that lead to anemission event, and is typically less than 1. In some embodiments, theluminescent quantum yield of a molecule described herein is between 0and about 0.001, between about 0.001 and about 0.01, between about 0.01and about 0.1, between about 0.1 and about 0.5, between about 0.5 and0.9, or between about 0.9 and 1. In some embodiments, a molecule isidentified by determining or estimating the luminescent quantum yield.

As used herein for single molecules luminescent intensity refers to thenumber of emitted photons per unit time that are emitted by a moleculewhich is being excited by delivery of a pulsed excitation energy. Insome embodiments, the luminescent intensity refers to the detectednumber of emitted photons per unit time that are emitted by a moleculewhich is being excited by delivery of a pulsed excitation energy, andare detected by a particular sensor or set of sensors.

In one aspect, the application provides a method of determining theluminescent lifetime of a single luminescent molecule comprising:providing the luminescent molecule in a target volume; delivering aplurality of pulses of an excitation energy to a vicinity of the targetvolume; and detecting a plurality of luminescences from the luminescentmolecule. In some embodiments, the method further comprises recording aplurality of time durations between each pair of pulses andluminescences and evaluating the distribution of the plurality of timedurations between each pair of pulses and luminescences.

C. Luminescently Labeled Nucleotides

In one aspect, methods and compositions described herein comprises oneor more luminescently labeled nucleotide. In certain embodiments, one ormore nucleotides comprise deoxyribose nucleosides. In some embodiments,all nucleotides comprises deoxyribose nucleosides. In certainembodiments, one or more nucleotides comprise ribose nucleosides. Insome embodiments, all nucleotides comprise ribose nucleosides. In someembodiments, one or more nucleotides comprise a modified ribose sugar orribose analog (e.g., a locked nucleic acid). In some embodiments, one ormore nucleotides comprise naturally occurring bases (e.g., cytosine,guanine, adenine, thymine, uracil). In some embodiments, one or morenucleotides comprise derivatives or analogs of cytosine, guanine,adenine, thymine, or uracil.

In certain embodiments, a method comprises the step of exposing apolymerase complex to a plurality of luminescently labeled nucleotides.In certain embodiments, a composition or device comprises a reactionmixture comprising a plurality of luminescently labeled nucleotides. Insome embodiments, the plurality of nucleotides comprises four types ofnucleotides. In some embodiments, the four types of nucleotides eachcomprise one of cytosine, guanine, adenine, and thymine. In someembodiments, the four types of nucleotides each comprise one ofcytosine, guanine, adenine, and uracil.

The term “nucleic acid,” as used herein, generally refers to a moleculecomprising one or more nucleic acid subunits. A nucleic acid may includeone or more subunits selected from adenosine (A), cytosine (C), guanine(G), thymine (T) and uracil (U), or variants thereof. In some examples,a nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA),or derivatives thereof. A nucleic acid may be single-stranded or doublestranded. A nucleic acid may be circular.

The term “nucleotide,” as used herein, generally refers to a nucleicacid subunit, which can include A, C, G, T or U, or variants or analogsthereof. A nucleotide can include any subunit that can be incorporatedinto a growing nucleic acid strand. Such subunit can be an A, C, G, T,or U, or any other subunit that is specific to one or more complementaryA, C, G, T or U, or complementary to a purine (i.e., A or G, or variantor analogs thereof) or a pyrimidine (i.e., C, T or U, or variant oranalogs thereof). A subunit can enable individual nucleic acid bases orgroups of bases (e.g., AA, TA, AT, GC, CG, CT, TC, GT, TG, AC, CA, oruracil-counterparts thereof) to be resolved.

A nucleotide generally includes a nucleoside and at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more phosphate (PO3) groups. A nucleotide can includea nucleobase, a five-carbon sugar (either ribose or deoxyribose), andone or more phosphate groups. Ribonucleotides are nucleotides in whichthe sugar is ribose. Deoxyribonucleotides are nucleotides in which thesugar is deoxyribose. A nucleotide can be a nucleoside monophosphate ora nucleoside polyphosphate. A nucleotide can be a deoxyribonucleosidepolyphosphate, such as, e.g., a deoxyribonucleoside triphosphate, whichcan be selected from deoxyadenosine triphosphate (dATP), deoxycytidinetriphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyuridinetriphosphate (dUTP) and deoxythymidine triphosphate (dTTP) dNTPs, thatinclude detectable tags, such as luminescent tags or markers (e.g.,fluorophores).

A nucleoside polyphosphate can have ‘n’ phosphate groups, where ‘n’ is anumber that is greater than or equal to 2, 3, 4, 5, 6, 7, 8, 9, or 10.Examples of nucleoside polyphosphates include nucleoside diphosphate andnucleoside triphosphate. A nucleotide can be a terminal phosphatelabeled nucleoside, such as a terminal phosphate labeled nucleosidepolyphosphate. Such label can be a luminescent (e.g., fluorescent orchemiluminescent) label, a fluorogenic label, a colored label, achromogenic label, a mass tag, an electrostatic label, or anelectrochemical label. A label (or marker) can be coupled to a terminalphosphate through a linker. The linker can include, for example, atleast one or a plurality of hydroxyl groups, sulfhydryl groups, aminogroups or haloalkyl groups, which may be suitable for forming, forexample, a phosphate ester, a thioester, a phosphoramidate or an alkylphosphonate linkage at the terminal phosphate of a natural or modifiednucleotide. A linker can be cleavable so as to separate a label from theterminal phosphate, such as with the aid of a polymerization enzyme.Examples of nucleotides and linkers are provided in U.S. Pat. No.7,041,812, which is entirely incorporated herein by reference.

D. Labels

In certain embodiments, the incorporated molecule is a luminescentmolecule, e.g., without attachment of a distinct luminescent marker.Typical nucleotide and amino acids are not luminescent, or do notluminesce within suitable ranges of excitation and emission energies. Incertain embodiments, the incorporated molecule comprises a luminescentmarker. In certain embodiments, the incorporated molecule is aluminescently labeled nucleotide. In certain embodiments, theincorporated molecule is a luminescently labeled amino acid orluminescently labeled tRNA. In some embodiments, a luminescently labelednucleotide comprises a nucleotide and a luminescent marker. In someembodiments, a luminescently labeled nucleotide comprises a nucleotide,a luminescent marker, and a linker. In some embodiments, the luminescentmarker is a fluorophore.

For nucleotide sequencing, certain combinations of luminescently labelednucleotides may be preferred. In some embodiments, at least one of theluminescently labeled nucleotides comprises a cyanine dye, or analogthereof. In some embodiments, at least one luminescently labelednucleotides comprises a rhodamine dye, or analog thereof. In someembodiments, at least one of the luminescently labeled nucleotidescomprises a cyanine dye, or analog thereof, and at least oneluminescently labeled nucleotides comprises a rhodamine dye, or analogthereof.

In certain embodiments, the luminescent marker is a dye selected fromTable FL-1. The dyes listed in Table FL-1 are non-limiting, and theluminescent markers of the application may include dyes not listed inTable FL-1. In certain embodiments, the luminescent markers of one ormore luminescently labeled nucleotides is selected from Table FL-1. Incertain embodiments, the luminescent markers of four or moreluminescently labeled nucleotides is selected from Table FL-1.

TABLE FL-1 Exemplary fluorophores. Fluorophores 5/6-Carboxyrhodamine 6G5-Carboxyrhodamine 6G 6-Carboxyrhodamine 6G 6-TAMRA Abberior Star 440SXPAbberior Star 470SXP Abberior Star 488 Abberior Star 512 Abberior Star520SXP Abberior Star 580 Abberior Star 600 Abberior Star 635 AbberiorStar 635P Abberior Star RED Alexa Fluor 350 Alexa Fluor 405 Alexa Fluor430 Alexa Fluor 480 Alexa Fluor 488 Alexa Fluor 514 Alexa Fluor 532Alexa Fluor 546 Alexa Fluor 555 Alexa Fluor 568 Alexa Fluor 594 AlexaFluor 610-X Alexa Fluor 633 Alexa Fluor 647 Alexa Fluor 660 Alexa Fluor680 Alexa Fluor 700 Alexa Fluor 750 Alexa Fluor 790 AMCA ATTO 390 ATTO425 ATTO 465 ATTO 488 ATTO 495 ATTO 514 ATTO 520 ATTO 532 ATTO 542 ATTO550 ATTO 565 ATTO 590 ATTO 610 ATTO 620 ATTO 633 ATTO 647 ATTO 647N ATTO655 ATTO 665 ATTO 680 ATTO 700 ATTO 725 ATTO 740 ATTO Oxa12 ATTO Rho101ATTO Rho11 ATTO Rho12 ATTO Rho13 ATTO Rho14 ATTO Rho3B ATTO Rho6G ATTOThio12 BD Horizon V450 BODIPY 493/501 BODIPY 530/550 BODIPY 558/568BODIPY 564/570 BODIPY 576/589 BODIPY 581/591 BODIPY 630/650 BODIPY650/665 BODIPY FL BODIPY FL-X BODIPY R6G BODIPY TMR BODIPY TR C5.5 C7CAL Fluor Gold 540 CAL Fluor Green 510 CAL Fluor Orange 560 CAL FluorRed 590 CAL Fluor Red 610 CAL Fluor Red 615 CAL Fluor Red 635 CascadeBlue CF350 CF405M CF405S CF488A CF514 CF532 CF543 CF546 CF555 CF568CF594 CF620R CF633 CF633-V1 CF640R CF640R-V1 CF640R-V2 CF660C CF660RCF680 CF680R CF680R-V1 CF750 CF770 CF790 Chromeo 642 Chromis 425NChromis 500N Chromis 515N Chromis 530N Chromis 550A Chromis 550C Chromis550Z Chromis 560N Chromis 570N Chromis 577N Chromis 600N Chromis 630NChromis 645A Chromis 645C Chromis 645Z Chromis 678A Chromis 678C Chromis678Z Chromis 770A Chromis 770C Chromis 800A Chromis 800C Chromis 830AChromis 830C Cy3 Cy3.5 Cy3B Cy5 DyLight 350 DyLight 405 DyLight 415-Co1DyLight 425Q DyLight 485-LS DyLight 488 DyLight 504Q DyLight 510-LSDyLight 515-LS DyLight 521-LS DyLight 530-R2 DyLight 543Q DyLight 550DyLight 554-R0 DyLight 554-R1 DyLight 590-R2 DyLight 594 DyLight 610-B1DyLight 615-B2 DyLight 633 DyLight 633-B1 DyLight 633-B2 DyLight 650DyLight 655-B1 DyLight 655-B2 DyLight 655-B3 DyLight 655-B4 DyLight 662QDyLight 675-B1 DyLight 675-B2 DyLight 675-B3 DyLight 675-B4 DyLight679-C5 DyLight 680 DyLight 683Q DyLight 690-B1 DyLight 690-B2 DyLight696Q DyLight 700-B1 DyLight 730-B1 DyLight 730-B2 DyLight 730-B3 DyLight730-B4 DyLight 747 DyLight 747-B1 DyLight 747-B2 DyLight 747-B3 DyLight747-B4 DyLight 755 DyLight 766Q DyLight 775-B2 DyLight 775-B3 DyLight775-B4 DyLight 780-B1 DyLight 780-B2 DyLight 780-B3 DyLight 800 DyLight830-B2 Dyomics-350 Dyomics-350XL Dyomics-360XL Dyomics-370XLDyomics-375XL Dyomics-380XL Dyomics-390XL Dyomics-405 Dyomics-415Dyomics-430 Dyomics-431 Dyomics-478 Dyomics-480XL Dyomics-481XLDyomics-485XL Dyomics-490 Dyomics-495 Dyomics-505 Dyomics-510XLDyomics-511XL Dyomics-520XL Dyomics-521XL Dyomics-530 Dyomics-547Dyomics-547P1 Dyomics-548 Dyomics-549 Dyomics-549P1 Dyomics-550Dyomics-554 Dyomics-555 Dyomics-556 Dyomics-560 Dyomics-590 Dyomics-591Dyomics-594 Dyomics-601XL Dyomics-605 Dyomics-610 Dyomics-615Dyomics-630 Dyomics-631 Dyomics-632 Dyomics-633 Dyomics-634 Dyomics-635Dyomics-636 Dyomics-647 Dyomics-647P1 Dyomics-648 Dyomics-648P1Dyomics-649 Dyomics-649P1 Dyomics-650 Dyomics-651 Dyomics-652Dyomics-654 Dyomics-675 Dyomics-676 Dyomics-677 Dyomics-678Dyomics-679P1 Dyomics-680 Dyomics-681 Dyomics-682 Dyomics-700Dyomics-701 Dyomics-703 Dyomics-704 Dyomics-730 Dyomics-731 Dyomics-732Dyomics-734 Dyomics-749 Dyomics-749P1 Dyomics-750 Dyomics-751Dyomics-752 Dyomics-754 Dyomics-776 Dyomics-777 Dyomics-778 Dyomics-780Dyomics-781 Dyomics-782 Dyomics-800 Dyomics-831 eFluor 450 Eosin FITCFluorescein HiLyte Fluor 405 HiLyte Fluor 488 HiLyte Fluor 532 HiLyteFluor 555 HiLyte Fluor 594 HiLyte Fluor 647 HiLyte Fluor 680 HiLyteFluor 750 IRDye 680LT IRDye 750 IRDye 800CW JOE LightCycler 640RLightCycler Red 610 LightCycler Red 640 LightCycler Red 670 LightCyclerRed 705 Lissamine Rhodamine B Napthofluorescein Oregon Green 488 OregonGreen 514 Pacific Blue Pacific Green Pacific Orange PET PF350 PF405PF415 PF488 PF505 PF532 PF546 PF555P PF568 PF594 PF610 PF633P PF647PQuasar 570 Quasar 670 Quasar 705 Rhoadmine 123 Rhodamine 6G Rhodamine BRhodamine Green Rhodamine Green-X Rhodamine Red ROX Seta 375 Seta 470Seta 555 Seta 632 Seta 633 Seta 650 Seta 660 Seta 670 Seta 680 Seta 700Seta 750 Seta 780 Seta APC-780 Seta PerCP-680 Seta R-PE-670 Seta646SeTau 380 SeTau 405 SeTau 425 SeTau 647 Square 635 Square 650 Square 660Square 672 Square 680 Sulforhodamine 101 TAMRA TET Texas Red TMR TRITCYakima Yellow Zenon Zy3 Zy5 Zy5.5 Zy7

In certain embodiments, the luminescent marker may be (Dye 101) or (Dye102), of formulae:

or an analog thereof. In some embodiments, each sulfonate or carboxylateis independently optionally protonated. In some embodiments, the dyesabove are attached to the linker or nucleotide by formation of an amidebond at the indicated point of attachment.

In certain embodiments, at least one type, at least two types, at leastthree types, or at least four of the types of luminescently labelednucleotides comprise a luminescent marker selected from the groupconsisting of 6-TAMRA, 5/6-Carboxyrhodamine 6G, Alex Fluor 546, AlexaFluor 555, Alexa Fluor 568, Alexa Fluor 610, Alexa Fluor 647, AberriorStar 635, ATTO 647N, ATTO Rhol4, Chromis 630, Chromis 654A, Chromeo 642,CF514, CF532, CF543, CF546, CF546, CF555, CF568, CF633, CF640R, CF660C,CF660R, CF680R, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Dyomics-530,Dyomics-547P1, Dyomics-549P1, Dyomics-550, Dyomics-554, Dyomics-555,Dyomics-556, Dyomics-560, Dyomics-650, Dyomics-680, DyLight 554-R1,DyLight 530-R2, DyLight 594, DyLight 635-B2, DyLight 650, DyLight655-B4, DyLight 675-B2, DyLight 675-B4, DyLight 680, HiLyte Fluor 532,HiLyte Fluor 555, HiLyte Fluor 594, LightCycler 640R, Seta 555, Seta670, Seta700, SeTau 647, and SeTau 665, or are of formulae (Dye 101) or(Dye 102), as described herein.

In some embodiments, at least one type, at least two types, at leastthree types, or at least four of the types of luminescently labelednucleotides comprise a luminescent marker selected from the groupconsisting of Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, AlexaFluor 594, Alexa Fluor 610, CF532, CF543, CF555, CF594, Cy3, DyLight530-R2, DyLight 554-R1, DyLight 590-R2, DyLight 594, and DyLight 610-B1,or are of formulae (Dye 101) or (Dye 102).

In some embodiments, a first and second type of luminescently labelednucleotide comprise a luminescent marker selected from the groupconsisting of Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, CF532,CF543, CF555, Cy3, DyLight 530-R2, and DyLight 554-R1, and a third andfourth type of luminescently labeled nucleotide comprise a luminescentlabel selected from the group consisting of Alexa Fluor 594, Alexa Fluor610, CF594, DyLight 590-R2, DyLight 594, and DyLight 610-B1, or are offormulae (Dye 101) or (Dye 102).

E. Linkers

A luminescent marker may be attached to the molecule directly, e.g., bya bond, or may be attached via a linker. In certain embodiments, thelinker comprises one or more phosphates. In some embodiments, anucleoside is connected to a luminescent marker by a linker comprisingone or more phosphates. In some embodiments, a nucleoside is connectedto a luminescent marker by a linker comprising three or more phosphates.In some embodiments, a nucleoside is connected to a luminescent markerby a linker comprising four or more phosphates.

In certain embodiments, a linker comprises an aliphatic chain. In someembodiments a linker comprises —(CH₂)_(n)—, wherein n is an integer from1 to 20, inclusive. In some embodiments, n is an integer from 1 to 10,inclusive. In certain embodiments, a linker comprises a heteroaliphaticchain. In some embodiments, a linker comprises a polyethylene glycolmoiety. In some embodiments, a linker comprises a polypropylene glycolmoiety. In some embodiments, a linker comprises —(CH₂CH₂O)_(n)—, whereinn is an integer from 1 to 20, inclusive. In some embodiments, a linkercomprises —(CH₂CH₂O)_(n)—, wherein n is an integer from 1 to 10,inclusive. In certain embodiments, a linker comprises —(CH₂CH₂O)₄—. Incertain embodiments, a linker comprises one or more arylenes. In someembodiments, a linker comprises one or more phenylenes (e.g.,para-substituted phenylene). In certain embodiments, a linker comprisesa chiral center. In some embodiments, a linker comprises proline, or aderivative thereof. In some embodiments, a linker comprises a prolinehexamer, or a derivative thereof. In some embodiments, a linkercomprises coumarin, or a derivative thereof. In some embodiments, alinker comprises naphthalene, or a derivative thereof. In someembodiments, a linker comprises anthracene, or a derivative thereof. Insome embodiments, a linker comprises a polyphenylamide, or a derivativethereof. In some embodiments, a linker comprises chromanone, or aderivative thereof. In some embodiments, a linker comprises4-aminopropargyl-L-phenylalanine, or a derivative thereof. In certainembodiments, a linker comprises a polypeptide.

In some embodiments, a linker comprises an oligonucleotide. In someembodiments, a linker comprises two annealed oligonucleotides. In someembodiments, the oligonucleotide or oligonucleotides comprisedeoxyribose nucleotides, ribose nucleotide, or locked ribosenucleotides. In certain embodiments, a linker comprises aphotostabilizer.

F. Sample Well Surface Preparation

In certain embodiments, a method of detecting one or luminescentlylabeled molecule is performed with the molecules confined in a targetvolume. In some embodiments, the target volume is a region within asample well (e.g., a nanoaperture). In certain embodiments, the samplewell comprises a bottom surface comprising a first material andsidewalls formed by a plurality of metal or metal oxide layers. In someembodiments, the first material is a transparent material or glass. Insome embodiments, the bottom surface is flat. In some embodiments, thebottom surface is a curved well. In some embodiments, the bottom surfaceincludes a portion of the sidewalls below the sidewalls formed by aplurality of metal or metal oxide layers. In some embodiments, the firstmaterial is fused silica or silicon dioxide. In some embodiments, theplurality of layers each comprise a metal (e.g., Al, Ti) or metal oxide(e.g., Al₂O₃, TiO₂, TiN).

G. Passivation

In some embodiments when one or more molecule or complex is immobilizedon a surface, it is desirable to passivate other surfaces of the deviceto prevent immobilization at an undesired location. In some embodiments,the molecule or complex is immobilized on a bottom surface of a samplewell and the sidewalls of the sample well are passivated. In someembodiments, the sidewalls are passivated by the steps of: depositing ametal or metal oxide barrier layer on the sidewall surfaces; andapplying a coating to the barrier layer. In some embodiments, the metaloxide barrier layer comprises aluminum oxide. In some embodiments, thestep of depositing comprises depositing the metal or metal oxide barrierlayer on the sidewall surfaces and the bottom surface. In someembodiments, the step of depositing further comprises etching metal ormetal oxide barrier layer off of the bottom surface.

In some embodiments, the barrier layer coating comprises phosphonategroups. In some embodiments, the barrier layer coating comprisesphosphonate groups with an alkyl chain. In some embodiments, the barrierlayer coating comprises a polymeric phosphonate. In some embodiments,the barrier layer coating comprises polyvinylphosphonic acid (PVPA). Insome embodiments, the barrier layer coating comprises phosphonate groupswith a substituted alkyl chain. In some embodiments, the alkyl chaincomprises one or more amides. In some embodiments, the alkyl chaincomprises one or more poly(ethylene glycol) chains. In some embodiments,the coating comprises phosphonate groups of formula:

wherein n is an integer between 0 and 100, inclusive, and

is hydrogen or a point of attachment to the surface. In some embodimentsn is an integer between 3 and 20, inclusive. In some embodiments, thebarrier layer coating comprises a mixture of different types ofphosphonate groups. In some embodiments, the barrier layer coatingcomprises a mixture of phosphonate groups comprising poly(ethyleneglycol) chains of different PEG weight.

In certain embodiments, the barrier layer comprises nitrodopa groups. Incertain embodiments, the barrier layer coating comprises groups offormula:

wherein R^(N) is an optionally substituted alkyl chain and

is hydrogen or a point of attachment to the surface. In someembodiments, R^(N) comprises a polymer. In some embodiments, R^(N)comprises a poly(lysine) or a poly(ethylene glycol). In someembodiments, the barrier layer comprises a co-polymer of poly(lysine)comprising lysine monomers, wherein the lysine monomers independentlycomprise PEG, nitrodopa groups, phosphonate groups, or primary amines.In certain embodiments, the barrier layer comprises a polymer of formula(P):

In some embodiments, X is —OMe, a biotin group, phosphonate, or silane.In some embodiments, each of i, j, k, and l is independently an integerbetween 0 and 100, inclusive.

H. Polymerase Immobilization

In some embodiments, when one or more molecule or complex is immobilizedon a surface the surface is functionalized to allow for attachment ofone or more of the molecules or complexes. In some embodiments, thefunctionalized surface is a bottom surface of a sample well. In certainembodiments, the functionalized surface comprises a transparent glass.In certain embodiments, the functionalized surface comprises fusedsilica or silicon dioxide. In some embodiments, the functionalizedsurface is functionalized with a silane. In some embodiments, thefunctionalized surface is functionalized with an ionically chargedpolymer. In some embodiments, the ionically charged polymer comprisespoly(lysine). In some embodiments, the functionalized surface isfunctionalized with poly(lysine)-graft-poly(ethylene glycol). In someembodiments, the functionalized surface is functionalized withbiotinylated bovine serum albumin (BSA).

In certain embodiments, the functionalized surface is functionalizedwith a coating comprising nitrodopa groups. In certain embodiments, thecoating comprises groups of formula:

wherein R^(N) is an optionally substituted alkyl chain and

is hydrogen or a point of attachment to the surface. In someembodiments, R^(N) comprises a polymer. In some embodiments, R^(N)comprises a poly(lysine) or a poly(ethylene glycol). In someembodiments, R^(N) comprises a biotinylated poly(ethylene glycol). Insome embodiments, the coating comprises a co-polymer of poly(lysine)comprising lysine monomers, wherein the lysine monomers independentlycomprise PEG, biotinylated PEG, nitrodopa groups, phosphonate groups, orsilanes. In certain embodiments, the coating comprises a polymer offormula (P):

In some embodiments, X is —OMe, a biotin group, phosphonate, or silane.In some embodiments, each of i, j, k, and l is independently an integerbetween 0 and 100, inclusive.

In some embodiments, the functionalized surface is functionalized with asilane comprising an alkyl chain. In some embodiments, thefunctionalized surface is functionalized with a silane comprising anoptionally substituted alkyl chain. In some embodiments, the surface isfunctionalized with a silane comprising a poly(ethylene glycol) chain.In some embodiments, the functionalized surface is functionalized with asilane comprising a coupling group. For example the coupling group maycomprise chemical moieties, such as amine groups, carboxyl groups,hydroxyl groups, sulfhydryl groups, metals, chelators, and the like.Alternatively, they may include specific binding elements, such asbiotin, avidin, streptavidin, neutravidin, lectins, SNAP-tags™ orsubstrates therefore, associative or binding peptides or proteins,antibodies or antibody fragments, nucleic acids or nucleic acid analogs,or the like. Additionally, or alternatively, the coupling group may beused to couple an additional group that is used to couple or bind withthe molecule of interest, which may, in some cases include both chemicalfunctional groups and specific binding elements. By way of example, acoupling group, e.g., biotin, may be deposited upon a substrate surfaceand selectively activated in a given area. An intermediate bindingagent, e.g., streptavidin, may then be coupled to the first couplinggroup. The molecule of interest, which in this particular example wouldbe biotinylated, is then coupled to the streptavidin.

In some embodiments, the functionalized surface is functionalized with asilane comprising biotin, or an analog thereof. In some embodiments, thesurface is functionalized with a silane comprising a poly(ethylene)glycol chain, wherein the poly(ethylene glycol) chain comprises biotin.In certain embodiments, the functionalized surface is functionalizedwith a mixture of silanes, wherein at least one type of silane comprisesbiotin and at least one type of silane does not comprise biotin. In someembodiments, the mixture comprises about 10 fold less, about 25 foldless, about 50 fold less, about 100 fold less, about 250 fold less,about 500 fold less, or about 1000 fold less of the biotinylated silanethan the silane not comprising biotin.

FIG. 10-3 depicts a non-limiting exemplary process for preparing thesample well surface from the fabricated chip (e.g., integrated device)to initiation of a sequencing reaction. The sample well is depicted witha bottom surface (unshaded rectangle) and sidewalls (shaded verticalrectangles). The sidewalls may be comprised of multiple layers (e.g.,Al, Al₂O₃, Ti, TiO₂, TiN). In step (a) the sidewalls are deposited witha barrier layer of Al₂O₃. The Al₂O₃ barrier layer is then coated, instep (b), with a PEG phosphonate groups, for example, by treating thesurface with one or more PEG-phosphonic acids. In step (c), the bottomsurface is functionalized, for example, with a mixture of PEG-silane andbiotinylated-PEG-silane. The ovals represent individual biotin groupswhich may provide sites for an attachment of a single molecule orcomplex, such as a polymerase complex. In step (d), a polymerase complexis attached to a biotin group on the bottom surface. The polymerase maybe attached by way of a binding agent, such as streptavidin, and abiotin tag on the polymerase complex. The polymerase complex may furthercomprise a template nucleic acid and primer (not shown). Step (e)depicts the initiation of a sequencing reaction by exposure of theimmobilized polymerase complex to luminescently labeled nucleotides.

I. Polymerases

The term “polymerase,” as used herein, generally refers to any enzyme(or polymerizing enzyme) capable of catalyzing a polymerizationreaction. Examples of polymerases include, without limitation, a nucleicacid polymerase, a transcriptase or a ligase. A polymerase can be apolymerization enzyme.

Embodiments directed towards single molecule nucleic acid extension(e.g., for nucleic acid sequencing) may use any polymerase that iscapable of synthesizing a nucleic acid complementary to a target nucleicacid molecule. In some embodiments, a polymerase may be a DNApolymerase, an RNA polymerase, a reverse transcriptase, and/or a mutantor altered form of one or more thereof.

Embodiments directed towards single molecule nucleic acid sequencing mayuse any polymerase that is capable of synthesizing a nucleic acidcomplementary to a target nucleic acid. Examples of polymerases include,but are not limited to, a DNA polymerase, an RNA polymerase, athermostable polymerase, a wild-type polymerase, a modified polymerase,E. coli DNA polymerase I, T7 DNA polymerase, bacteriophage T4 DNApolymerase φ²⁹ (psi29) DNA polymerase, Taq polymerase, Tth polymerase,Tli polymerase, Pfu polymerase, Pwo polymerase, VENT polymerase,DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Ssopolymerase, Poc polymerase, Pab polymerase, Mth polymerase, ES4polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tmapolymerase, Tca polymerase, Tih polymerase, Tfi polymerase, Platinum Taqpolymerases, Tbr polymerase, Tfl polymerase, Tth polymerase, Pfutubopolymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bstpolymerase, Sac polymerase, Klenow fragment, polymerase with 3′ to 5′exonuclease activity, and variants, modified products and derivativesthereof. In some embodiments, the polymerase is a single subunitpolymerase. Non-limiting examples of DNA polymerases and theirproperties are described in detail in, among other places, DNAReplication 2nd edition, Kornberg and Baker, W. H. Freeman, New York,N.Y. (1991).

Upon base pairing between a nucleobase of a target nucleic acid and thecomplementary dNTP, the polymerase incorporates the dNTP into the newlysynthesized nucleic acid strand by forming a phosphodiester bond betweenthe 3′ hydroxyl end of the newly synthesized strand and the alphaphosphate of the dNTP. In examples in which the luminescent tagconjugated to the dNTP is a fluorophore, its presence is signaled byexcitation and a pulse of emission is detected during or after the stepof incorporation. For detection labels that are conjugated to theterminal (gamma) phosphate of the dNTP, incorporation of the dNTP intothe newly synthesized strand results in release the beta and gammaphosphates and the detection label, which is free to diffuse in thesample well, resulting in a decrease in emission detected from thefluorophore.

In some embodiments, the polymerase is a polymerase with highprocessivity. However, in some embodiments, the polymerase is apolymerase with reduced processivity. Polymerase processivity generallyrefers to the capability of a polymerase to consecutively incorporatedNTPs into a nucleic acid template without releasing the nucleic acidtemplate.

In some embodiments, the polymerase is a polymerase with low 5′-3′exonuclease activity and/or 3′-5′ exonuclease. In some embodiments, thepolymerase is modified (e.g., by amino acid substitution) to havereduced 5′-3′ exonuclease activity and/or 3′-5′ activity relative to acorresponding wild-type polymerase. Further non-limiting examples of DNApolymerases include 9° Nm™ DNA polymerase (New England Biolabs), and aP680G mutant of the Klenow exo-polymerase (Tuske et al. (2000) JBC275(31):23759-23768). In some embodiments, a polymerase having reducedprocessivity provides increased accuracy for sequencing templatescontaining one or more stretches of nucleotide repeats (e.g., two ormore sequential bases of the same type).

Embodiments directed toward single molecule RNA extension (e.g., for RNAsequencing) may use any reverse transcriptase that is capable ofsynthesizing complementary DNA (cDNA) from an RNA template. In suchembodiments, a reverse transcriptase can function in a manner similar topolymerase in that cDNA can be synthesized from an RNA template via theincorporation of dNTPs to a reverse transcription primer annealed to anRNA template. The cDNA can then participate in a sequencing reaction andits sequence determined as described above and elsewhere herein. Thedetermined sequence of the cDNA can then be used, via sequencecomplementarity, to determine the sequence of the original RNA template.Examples of reverse transcriptases include Moloney Murine Leukemia Virusreverse transcriptase (M-MLV), avian myeloblastosis virus (AMV) reversetranscriptase, human immunodeficiency virus reverse transcriptase(HIV-1) and telomerase reverse transcriptase.

The processivity, exonuclease activity, relative affinity for differenttypes of nucleic acid, or other property of a nucleic acid polymerasecan be increased or decreased by one of skill in the art by mutation orother modification relative to a corresponding wild-type polymerase.

J. Lifetime Measurements

Lifetime measurements may be performed using one excitation energywavelength to excite a marker in a sample well. A combination of markershaving distinct lifetimes are selected to distinguish among theindividual markers based on the lifetime measurements. Additionally, thecombination of markers are able to reach an excited state whenilluminated by the excitation source used. Any set or number of suitablemarkers may be used that have distinguishable lifetimes. For example,three, four or six different markers may be used. The luminescentmarkers may excite at the same excitation wavelength or two or fourdifferent excitation wavelengths.

A pulsed excitation source may be one of the pulsed excitation sourcesusing the techniques described above. In some instances, the pulsedexcitation source may be a semiconductor laser diode configured to emitpulses through direct modulated electrical pumping of the laser diode.The power of the pulses is less than 20 dB of the pulse peak power atapproximately 250 picoseconds after the peak. The time interval for eachexcitation pulse is in the range of 20-200 picoseconds. The timeduration between each excitation pulse is in the range of 1-50nanoseconds. A schematic of how example measurements may be performed isshown in FIG. 10-4, which illustrates the timing of excitation pulses10-401 generated at regular intervals. The bottom row of FIG. 10-4indicates what label is present when an excitation pulse 10-401 arrives.On occasion, there will be no label present. Moreover, the middle row ofFIG. 10-4 illustrates the emission probability distribution 10-402 forthe label that is present. Also illustrated in the middle row is arectangle 10-403 indicating the detection of a photon from the label. Asshown in FIG. 10-4, there are times when a photon is not emitted or thephoton that is emitted does not get detected due to loss or detectorinefficiency.

The sensor for each pixel has at least one photosensitive region per apixel. In some embodiments, a sensor chip may include a single sensorregion per pixel. The photosensitive region may have dimensions of 5microns by 5 microns. Photons are detected within time intervals of whenthey reach the sensor. Increasing the number of time bins may improveresolution of the recorded histogram of photons collected over a seriesof time bins and improve differentiation among different luminescentmarkers. In some embodiments, focusing elements may be integrated withthe sensor in order to improve collection of photons emitted by a markerin an associated sample well. Such focusing elements may include aFresnel lens 10-500 as shown in FIG. 10-5. When the sensor is configuredto detect a particular wavelength, the four luminescent markers may emitluminescence similar to the particular wavelength. Alternatively, thefour luminescent markers may emit luminescence at different wavelengths.

In some embodiments, a sample object may be labeled with one of aplurality of different types of markers, each associated with a mutuallyexclusive set of sample objects. Each of the plurality of markers emitsemission energy with a different lifetime. When the sensor is configuredto detect a particular wavelength, the plurality markers may emitemission energy similar to the particular wavelength. Alternatively theplurality of markers may emit emission energy at different wavelengths.Techniques described herein for determining a lifetime for a marker maybe used. In response to a pulse of excitation energy from theexcitations source, one of the plurality of markers tagged to the samplemay emit a photon. The time of the photon after the excitation pulse isrecorded. Repeated pulses of excitation energy may produce multiplephoton emission events which are then used to determine a lifetime forthe marker. The determined lifetime may then be used to identify themarker in the sample well from among the plurality of markers.

An example set of four luminescent markers that are distinguishablebased on lifetime measurements are ATRho14, Cy5, AT647N, and CF633 asshown by the plot in FIG. 10-6. These four markers have varyinglifetimes and produce distinguishing histograms when at least four timebins are used. FIG. 10-7 outlines a signal profile for each of thesemarkers across 16 time bins. The signal profile is normalized for eachmarker. The time bins vary in time interval in order to provide a uniquesignal profile for each of the markers. As illustrated in FIG. 10-7, asensor having 16 time bins defined by time boundaries with variablespacing can be used to distinguish among ATTORho14, Cy5, ATTO647N, andCF633 by the distribution of photon counts across one or more of the 16time bins. For example, the sensor detects 11% of total light detectedby marker ATTORho14 between time boundaries 0.692 ns and 0.792 ns. Asanother example, the sensor detects 10% of total light detected frommarker CF633 between time boundaries 1.991 ns and 2.507 ns. In thismanner, each marker may be distinguished by the total amount of light inone or more time bins. FIGS. 10-8 and 10-9 illustrates signal profiles,both continuous and discrete, respectively, of another exemplary set ofmarkers, ATTO Rho14, D650, ST647, and CF633, that are distinguishablebased on lifetime measurements. Other sets of markers include ATTORho14, C647, ST647, CF633; Alexa Fluor647, B630, C640R, CF633; and ATTORho14, ATTO 647N, AlexaFluor647, CF633.

K. Spectral-Lifetime Measurements

Lifetime measurements may be combined with spectral measurements of oneor more luminescent markers. Spectral measurements may depend on thewavelength of emission energy for individual markers and are capturedusing at least two sensor regions per pixel. The sensor is configured todetect spectral properties of the emission energy emitted from a samplewell. An exemplary structure of the integrated device includes pixelsthat each have a sensor with two distinct regions, each regionconfigured to detect a different wavelength. Some markers may havespectra that substantially overlap and/or have peak emission wavelengthsthat differ by approximately 5 nm or less, for example, and aredifficult to distinguish based on spectral detection techniques alone.However, these markers may have varying lifetimes, and techniques forperforming lifetime measurements may be used to distinguish among themarkers.

Combining both lifetime measurements with spectral measurements may beperformed using one excitation energy wavelength to excite a marker in asample well, although more than one excitation energy wavelengths may beused, in some embodiments. A combination of markers is selected havingat least two distinct emission wavelengths where the markers emitting ata wavelength have distinct lifetimes are selected to distinguish amongthe individual markers based on the lifetime and spectral measurements.Additionally, the combination of markers is selected to be able to reachan excited state when illuminated by the excitation source used. Any setor number of suitable markers may be used that have different emissionwavelengths and/or different lifetimes.

The excitation source is a pulsed excitation source, and may be one ofthe excitation sources using the techniques described above. In someinstances, the pulsed excitation source may be a semiconductor laserdiode configured to emit pulses through direct modulated electricalpumping of the laser diode. The power of the pulses are less than 20 dBthe peak power after 250 picoseconds after the peak. The time durationfor each excitation pulse is in the range of 20-200 picoseconds. Thetime interval between each excitation pulse is in the range of 1-50nanoseconds. A schematic of how example measurements may be performed isshown in FIG. 10-10. In some embodiments, the excitation source providesexcitation energy having a wavelength of approximately 640 nm. In someembodiments, the excitation source provides excitation energy having awavelength in the range of approximately 515 nm to 535 nm.

The sensor is configured to detect both temporal and spectral propertiesof the emission energy from a plurality of markers. The sensor for eachpixel has at least two photosensitive regions per pixel. In someembodiments there are two photosensitive regions per a pixel. In otherembodiments, there are four photosensitive regions per a pixel. Eachphotosensitive region is configured to detect a different wavelength orrange of wavelengths. Photons are detected within time intervals of whenthey reach the sensor. Increasing the number of time bins may improveresolution of the recorded histogram of photons collected over a seriesof time bins and improve differentiation among different luminescentmarkers by their individual lifetimes. In some embodiments, there aretwo time bins per a region of the sensor. In other embodiments, thereare four time bins per a region of the sensor.

In some embodiments, a sensor includes two sub-sensors that are used todetect four different markers. A first sub-sensor may be associated withthe first emission wavelength emitted by two of the markers. The firstsub-sensor may also be associated with two distinct lifetimes from thetwo markers that emit at the first emission wavelength. A secondsub-sensor may be associated with the second wavelength emitted by theother two markers. The second sub-sensor may also be associated with twodistinct lifetimes from the two markers that emit at the second emissionwavelength. Differentiation among the four luminescent markers may occurbased on a combination of detected wavelength and detected lifetime.Each of the two sub-sensors may detect a single photon of the emissionenergy emitted from the marker.

An example set of four luminescent markers that are distinguishablebased on lifetime measurements are ATTO Rho14, AS635, Alexa Fluor647,and ATTO 647N. These four markers have two that emit at one similarwavelength and another similar wavelength. Within each pair of markersthat emit at a similar wavelength, the pair of markers have differentlifetimes and produce distinguishing histograms when at least four timebins are used. In this example, ATTO Rho14 and AS635 emit similarluminescence wavelengths and have distinct lifetimes. Alexa Fluor 647and ATTO 647N emit similar luminescence wavelengths, different from thewavelengths emitted by ATTO Rho 14 and AS635, and have distinctlifetimes. FIG. 10-11 shows a plot lifetime as a function of emissionwavelength for this set of markers to illustrate how each of thesemarkers is distinguishable based on a combination of lifetime andemission wavelength. As shown in FIG. 10-11, ATTO Rho14 may emit near645 nm, AS635 may emit near 653 nm, Alexa Fluor 647 may emit near 665nm, and ATTO Fluor 647N may emit near 669 nm. AS635 and ATTO 647N havesimilar lifetimes at approximately 1.2 ns, and ATTO Rho14 and AlexaFluor 647 have lower lifetimes than both AS635 and ATTO 647. Thesemarkers can be distinguished based on their characteristic emissionwavelength and emission lifetime. For example, ATTO 647N and Alexa Fluor647 have similar peak emission wavelengths, but have distinguishablelifetimes. AS635 and ATTO 647N have similar lifetimes, but havesubstantially different peak emission wavelengths.

The markers may excite at the same excitation wavelength or two or moredifferent excitation wavelengths. When the sensor is configured todetect two wavelengths of emission energy, two of the four markers mayemit at a first wavelength while the other two markers may emit at asecond wavelength. Alternatively, when the sensor is configured todetect four wavelengths of emission, each marker may emit at a differentwavelength and/or may have different emission probability densities andlifetimes. FIG. 10-12 shows a plot of power as a function of wavelengthfor ATT Rho14, Alexa Fluor 647, and ATT) 647N.

FIG. 10-13 shows plots of fluorescence signal over time for each one ofthese markers when present in a sample well with a diameter of 135 nm toillustrate the different time decay, and thus, lifetimes, for thesemarkers.

FIG. 10-14 illustrates the signal profile for these markers across foursensor regions and each region captures four time bins. The signalprofiles are normalized and are used to distinguish among the differentmarkers by the relative number of photons captured by a photosensitiveregion for each of the four time bins. Light of a first emissionwavelength, emitted from a first marker (Alexa Fluor 647), is directedtowards a first sensor region. However, the directionality of the lightis not perfect and some of the light is detected by a second sensorregion, a third sensor region and a fourth sensor region. Thus, lightemitted from the first marker is associated with a first sensor detectorsignal, as illustrated in the middle schematic of FIG. 10-14 by thestatistics for percentage of total light detected by the four sensorregions over four time bins. In the second time bin between 1.184 ns and2.069 ns, the first sensor region detects 4.2% of the total detectedlight from the first marker, the second sensor region detects 11.7% ofthe total detected light from the first marker, the third sensor regiondetects 18.6% of the total detected light from the first marker, and thefourth sensor region detects 14.6% of the total detected light from thefirst marker. This detected sensor pattern for one or more time binsconstitutes a first detection signal associated with the first marker.FIG. 10-14 shows sensor and time bin patterns for ATRho14 and ATTO647Nmarkers, which results in distinguishable detection signals among atleast these three markers. For example, the second sensor detects 12.3%of the total light from marker ATTORho14 in the first time bin between0.25 ns and 1.184 ns. In this manner, each marker may be distinguishedbased on its emission spectra and lifetime.

Other sets of four fluorophores for such spectral-lifetime measurementsare ATRho14, D650, ST647, CF633; ATTO Rho14, C647, ST647, CF633; AlexaFluor 647, B630, C640R, CF633; and ATTO Rho 14, ATTO 647N, Alexa Fluor647, CF633. FIG. 10-15 shows a plot of the signal profile of intensityover time for ATRho14, D650, ST647, and C633. FIG. 10-16 illustrates thesignal profile for ATRho14.

L. Lifetime-Excitation Energy Measurements

Lifetime measurements combined with using at least two excitation energywavelengths may be used to distinguish among multiple markers. Somemarkers may excite when one excitation wavelength is used and notanother. A combination of markers having distinct lifetimes is selectedfor each excitation wavelength to distinguish among the individualmarkers based on the lifetime measurements. In this embodiment, a sensorchip may be configured to have each pixel with a sensor having oneregion and the instrument may be configured to provide at least twoexcitation energy wavelengths, such as by electrically modulated pulseddiode lasers with temporal interleaving.

The markers may excite at different excitation wavelengths. When thelight source is configured to deliver two wavelengths of excitationenergy, some markers may excite at a first wavelength of excitation andnot excite at a second wavelength of excitation, while the other markersexcite at a second wavelength of excitation and not substantially exciteas a first wavelength of excitation. In some embodiments, two markersexcite at the first excitation wavelength and do not substantiallyexcite at the second excitation wavelength, but emit at differentemission probability densities and lifetimes. Two different markersexcite at the second excitation wavelength and do not substantiallyexcite at the first excitation wavelength, but have different emissionprobability densities and lifetimes.

The excitation source is a combination of at least two excitationenergies. The excitation source is a pulsed excitation source and may beone or more of the excitation sources using the techniques describedabove. In some instances, the pulsed excitation source may be twosemiconductor laser diode configured to emit pulses through directmodulated electrical pumping of the laser diode. The power of the pulsesare 20 dB less than the pulse peak power at 250 picoseconds after thepeak. The time interval for each excitation pulse is in the range of20-200 picoseconds. The time interval between each excitation pulse isin the range of 1-50 nanoseconds. One excitation wavelength is emittedper a pulse and by knowing the excitation wavelength a subset of markerswith distinct lifetimes is uniquely identified. In some embodiments,pulses of excitation alternate among the different wavelengths. Forexample, when two excitation wavelengths are used subsequent pulsesalternate between one wavelength and the other wavelength. A schematicof how example measurements may be performed is shown in FIG. 10-17. Anysuitable technique for combining multiple excitation sources andinterleaving pulses having different wavelengths may be used.

The sensor for each pixel has at least one photosensitive region per apixel. The photosensitive region may have dimensions of 5 microns by 5microns. Photons are detected within time intervals of when they reachthe sensor. Increasing the number of time bins may improve resolution ofthe recorded histogram of photons collected over a series of time binsand improve differentiation among different luminescent markers. Thesensor has at least two time bins. In some embodiments, the sensor mayhave four time bins to identify the lifetime of emission energy from amarker.

An example set of four luminescent markers that are distinguishablebased on lifetime measurements are Alexa Fluor 546, Cy3B, Alexa Fluor647, and ATTO 647N. As shown in FIG. 10-18, Alexa Fluor 546 and Cy3Bexcite at one wavelength, such as 532 nm, and have distinct lifetimes.Alexa Fluor 647 and ATTO 647N excite at another wavelength, 640 nm, andhave distinct lifetimes as shown in FIG. 10-19. For example, curves forATTO 647N and Alexa Fluor 647 are shown in FIG. 10-19 that illustratethese two markers having different lifetimes. Distinguishable normalizedsignal profiles across 16 time bins for ATTO647N and CF633, which areboth excited at 640 nm, are shown in FIG. 10-20. For example, the sensordetects 12% of total light detected from marker ATTO 647N between timeboundaries 0.629 ns and 0.997 ns. By detecting a photon after a knownexcitation wavelength, one of these two pairs of markers may bedetermined based on the previous excitation wavelength and each markerfor a pair is identified based on lifetime measurements.

M. Spectral Measurements

One or more sensors are configured to detect spectral properties byincluding one or more layers of the assay chip or sensor chip configuredto sort the wavelengths before they reach the sensor. One or more layersmay include at least one structural component configured to spectrallysort emission energy emitted from a sample well. The at least onestructural component may include an offset Fresnel lens, a blazed phasegrating, or any other suitable structure configured to provide desireddirectivity for spectrally distribution emission energy. A pixel of asensor chip may include multiple sub-sensors configured to detect aspectral distribution of emission energy directed from a correspondingsample well on an assay chip. The configuration of the sensor chip andthe multiple sub-sensors on the sensor chip may be sized, shaped, andarranged to sufficiently distinguish among different markers used tolabel a sample. Differentiation among luminescent markers may occurbased on the detected wavelength.

In some embodiments, the sensor may be segmented into four sub-sensorsto detect the four different luminescent markers. The sensor may besized and positioned in any suitable way to capture the emitted emissionenergy from the sample well.

In some embodiments, each sub-sensor is associated with a differentluminescence wavelength. Light of a first luminescence wavelength,emitted from a first luminescent marker (e.g., Alexa Fluor 555), isdirected towards a first sub-sensor. However, the directionality of thelight is not perfect and some of the light is detected by a secondsub-sensor, a third sub-sensor and a fourth sub-sensor. Thus, lightemitted from the first luminescent marker is associated with a firstsensor detector signal, as illustrated in FIG. 10-21. When photons arecollected during the integration time of the sensors, the firstsub-sensor detects 42% of the total light detected from the firstluminescent marker, the second sub-sensor detects 39% of the total lightdetected from the first luminescent marker, the third sub-sensor detects15% of the total light detected from the first luminescent marker, andthe fourth sub-sensor detects 4% of the total light detected from thefirst luminescent marker. This detected sub-sensor pattern constitutes afirst detection signal associated with the first luminescent marker.Different sub-sensor patterns are associated with different luminescencewavelengths from different luminescent markers which results indistinguishable detection signals, as illustrated in FIG. 10-21.

Similarly, the second sub-sensor is associated with a secondluminescence wavelength, the third sub-sensor is associated with a thirdluminescence wavelength, and the fourth sub-sensor is associated with afourth luminescence wavelength. In this manner, exemplary markers thatare distinguishable based on emission energy are Alexa Fluor 555, AlexaFluor 568, Alexa Fluor 647, and Alexa Fluor 660, which have emissionspectra shown in FIG. 10-22.

VII. COMPUTING DEVICE

FIG. 10-23 illustrates an example of a suitable computing systemenvironment 1000 on which embodiments may be implemented. For example,computing device 2-130 of FIG. 2-1 may be implemented according to thecomputing system environment 1000. Additionally, the computing systemenvironment 1000 may act as a control system that is programmed tocontrol the instrument to perform an assay. For example, the controlsystem may control the excitation source to emit and direct lighttowards the sample wells of the assay chip; control the sensors to allowdetection of emission light from one or more samples in the samplewells; and analyze signals from the sensors to identify, e.g., byanalyzing the spatial distribution of the emission energy, the samplepresent in a sample well. The computing system environment 1000 is onlyone example of a suitable computing environment and is not intended tosuggest any limitation as to the scope of use or functionality of theinvention. Neither should the computing environment 1000 be interpretedas having any dependency or requirement relating to any one orcombination of components illustrated in the exemplary operatingenvironment 1000.

Embodiments are operational with numerous other general purpose orspecial purpose computing system environments or configurations.Examples of well-known computing systems, environments, and/orconfigurations that may be suitable for use with the invention include,but are not limited to, personal computers, server computers, hand-heldor laptop devices, multiprocessor systems, microprocessor-based systems,set top boxes, programmable consumer electronics, network PCs,minicomputers, mainframe computers, distributed computing environmentsthat include any of the above systems or devices, and the like.

The computing environment may execute computer-executable instructions,such as program modules. Generally, program modules include routines,programs, objects, components, data structures, etc. that performparticular tasks or implement particular abstract data types. Theinvention may also be practiced in distributed computing environmentswhere tasks are performed by remote processing devices that are linkedthrough a communications network. In a distributed computingenvironment, program modules may be located in both local and remotecomputer storage media including memory storage devices.

With reference to FIG. 10-23, an exemplary system for implementing theinvention includes a general purpose computing device in the form of acomputer 1010. Components of computer 1010 may include, but are notlimited to, a processing unit 1020, a system memory 1030, and a systembus 1021 that couples various system components including the systemmemory to the processing unit 1020. The system bus 1021 may be any ofseveral types of bus structures including a memory bus or memorycontroller, a peripheral bus, and a local bus using any of a variety ofbus architectures. By way of example, and not limitation, sucharchitectures include Industry Standard Architecture (ISA) bus, MicroChannel Architecture (MCA) bus, Enhanced ISA (EISA) bus, VideoElectronics Standards Association (VESA) local bus, and PeripheralComponent Interconnect (PCI) bus also known as Mezzanine bus.

Computer 1010 typically includes a variety of computer readable media.Computer readable media can be any available media that can be accessedby computer 1010 and includes both volatile and nonvolatile media,removable and non-removable media. By way of example, and notlimitation, computer readable media may comprise computer storage mediaand communication media. Computer storage media includes both volatileand nonvolatile, removable and non-removable media implemented in anymethod or technology for storage of information such as computerreadable instructions, data structures, program modules or other data.Computer storage media includes, but is not limited to, RAM, ROM,EEPROM, flash memory or other memory technology, CD-ROM, digitalversatile disks (DVD) or other optical disk storage, magnetic cassettes,magnetic tape, magnetic disk storage or other magnetic storage devices,or any other medium which can be used to store the desired informationand which can accessed by computer 1010. Communication media typicallyembodies computer readable instructions, data structures, programmodules or other data in a modulated data signal such as a carrier waveor other transport mechanism and includes any information deliverymedia. The term “modulated data signal” means a signal that has one ormore of its characteristics set or changed in such a manner as to encodeinformation in the signal. By way of example, and not limitation,communication media includes wired media such as a wired network ordirect-wired connection, and wireless media such as acoustic, RF,infrared and other wireless media. Combinations of the any of the aboveshould also be included within the scope of computer readable media.

The system memory 1030 includes computer storage media in the form ofvolatile and/or nonvolatile memory such as read only memory (ROM) 1031and random access memory (RAM) 1032. A basic input/output system 1033(BIOS), containing the basic routines that help to transfer informationbetween elements within computer 1010, such as during start-up, istypically stored in ROM 1031. RAM 1032 typically contains data and/orprogram modules that are immediately accessible to and/or presentlybeing operated on by processing unit 1020. By way of example, and notlimitation, FIG. 10-23 illustrates operating system 1034, applicationprograms 1035, other program modules 1036, and program data 1037.

The computer 1010 may also include other removable/non-removable,volatile/nonvolatile computer storage media. By way of example only,FIG. 10-23 illustrates a hard disk drive 1041 that reads from or writesto non-removable, nonvolatile magnetic media, a magnetic disk drive 1051that reads from or writes to a removable, nonvolatile magnetic disk1052, and an optical disk drive 1055 that reads from or writes to aremovable, nonvolatile optical disk 1056 such as a CD ROM or otheroptical media. Other removable/non-removable, volatile/nonvolatilecomputer storage media that can be used in the exemplary operatingenvironment include, but are not limited to, magnetic tape cassettes,flash memory cards, digital versatile disks, digital video tape, solidstate RAM, solid state ROM, and the like. The hard disk drive 1041 istypically connected to the system bus 1021 through an non-removablememory interface such as interface 1040, and magnetic disk drive 1051and optical disk drive 1055 are typically connected to the system bus1021 by a removable memory interface, such as interface 1050.

The drives and their associated computer storage media discussed aboveand illustrated in FIG. 10, provide storage of computer readableinstructions, data structures, program modules and other data for thecomputer 1010. In FIG. 10-23, for example, hard disk drive 1041 isillustrated as storing operating system 1044, application programs 1045,other program modules 1046, and program data 1047. Note that thesecomponents can either be the same as or different from operating system1034, application programs 1035, other program modules 1036, and programdata 1037. Operating system 1044, application programs 1045, otherprogram modules 1046, and program data 1047 are given different numbershere to illustrate that, at a minimum, they are different copies. A usermay enter commands and information into the computer 1010 through inputdevices such as a keyboard 1062 and pointing device 1061, commonlyreferred to as a mouse, trackball or touch pad. Other input devices (notshown) may include a microphone, joystick, game pad, satellite dish,scanner, or the like. These and other input devices are often connectedto the processing unit 1020 through a user input interface 1060 that iscoupled to the system bus, but may be connected by other interface andbus structures, such as a parallel port, game port or a universal serialbus (USB). A monitor 1091 or other type of display device is alsoconnected to the system bus 1021 via an interface, such as a videointerface 1090. In addition to the monitor, computers may also includeother peripheral output devices such as speakers 1097 and printer 1096,which may be connected through a output peripheral interface 1095.

The computer 1010 may operate in a networked environment using logicalconnections to one or more remote computers, such as a remote computer1080. The remote computer 1080 may be a personal computer, a server, arouter, a network PC, a peer device or other common network node, andtypically includes many or all of the elements described above relativeto the computer 1010, although only a memory storage device 1081 hasbeen illustrated in FIG. 10-23. The logical connections depicted in FIG.10-23 include a local area network (LAN) 1071 and a wide area network(WAN) 1073, but may also include other networks. Such networkingenvironments are commonplace in offices, enterprise-wide computernetworks, intranets and the Internet.

When used in a LAN networking environment, the computer 1010 isconnected to the LAN 1071 through a network interface or adapter 1070.When used in a WAN networking environment, the computer 1010 typicallyincludes a modem 1072 or other means for establishing communicationsover the WAN 1073, such as the Internet. The modem 1072, which may beinternal or external, may be connected to the system bus 1021 via theuser input interface 1060, or other appropriate mechanism. In anetworked environment, program modules depicted relative to the computer1010, or portions thereof, may be stored in the remote memory storagedevice. By way of example, and not limitation, FIG. 10-23 illustratesremote application programs 1085 as residing on memory device 1081. Itwill be appreciated that the network connections shown are exemplary andother means of establishing a communications link between the computersmay be used.

IX. FABRICATION STEPS

The above integrated device may be fabricated in any suitable way. Whatfollows is a description of the fabrication of various components of theintegrated device, which may be combined in any way with techniquesknown in the art, to create a suitable integrated device.

A. Nanoaperture Fabrication Process

A sample well (e.g., nanoaperature) may be fabricated in any suitableway. In some embodiments, a sample well may be fabricated using standardphotolithography processes and etching techniques. A sample well may beformed in a layer having a metal (e.g., Al, TiN) or any suitablematerial compatible with photolithography processing. FIG. 11-1illustrates an exemplary method for fabricating a sample well of anintegrated device. Layer 11-112 forms a sample well and may include ametal such as Al or TiN. Layer 11-110 may act as a dielectric layer andmay be formed from any suitable dielectric substrate, such as SiO₂ orsilicon nitride. One step of the method includes depositing layer 11-112directly on substrate 11-110. In some embodiments, additional layers maybe deposited between layer 11-112 and layer 11-110. Layer 11-112 may bedeposited at any suitable thickness, and in some embodiments, thethickness may determine the height of the resulting sample well. Thethickness of layer 11-112 may be approximately 50 nm, approximately 100nm, or approximately 150 nm. An anti-reflection coating (ARC) 11-122 isthen deposited on top of layer 11-112. Etch mask 11-120 (e.g.,photoresist etch mask) is deposited on ARC 11-122. Conventionalphotolithographic techniques are used to pattern a hole in etch mask11-120 and ARC 11-122. The hole patterned by etch mask 11-120 may have adiameter of approximately 50 nm, approximately 100 nm, or approximately150 nm. The hole pattern is then transferred to underlying layer 11-112using an etch, for example, reactive ion etching techniques, to form thesample well. Etching may stop at the surface of layer 11-110, or etchingmay create a divot in layer 11-110 under the hole in layer 11-112.Conventional techniques are used to strip etch mask 11-120 and ARC11-122 off of layer 11-112. The sample well may have a diameter of tapproximately 50 nm, approximately 100 nm, or approximately 150 nm.

Alternatively, a sample well may be fabricated using standardphotolithography processes and lift-off techniques. FIG. 11-2illustrates an exemplary method of forming a sample well using lift-offtechniques. Sample well is formed in layer 11-212, which may include ametal (e.g., Al, Au, Cr). Layer 11-212 is formed over substrate layer11-210, which may include any suitable material such as a dielectric(e.g., SiO₂). Deposition of layer 11-212 may occur separately from andafter the photolithographic process. The first step in the lift-offfabrication process shown in FIG. 11-2 may involve depositinganti-reflection coating (ARC) 11-222 on substrate 11-210 followed byphotoresist etch mask 11-220 directly on top of substrate 11-210.Conventional photolithographic techniques are used to pattern thephotoresist such that a pillar 11-230 of resist is left behind. Thepillar may have any suitable size and shape that may correspond to aresulting sample well. The pillar may have a diameter of approximately50 nm, approximately 100 nm, or approximately 150 nm. Such techniquesmay include dissolving the resist and the ARC layer around the pillaroff of the substrate. The following step may involve depositing layer11-212 directly on top of the pillar of resist and the substrate,creating a capped pillar. In other embodiments, additional layers may bedeposited before or after deposition of layer 11-212. As a non-limitingexample, TiN may be deposited on layer 11-212 formed of Al, optionallyfollowed by a deposition of Al₂O₃. Layer 11-212 may be deposited at anysuitable thickness, and in some embodiments may have a thickness ofapproximately 50 nm, approximately 100 nm, or approximately 150 nm. Toform the sample well, the capped pillar may be stripped by a solvent inthe case that photoresist is used or by selective etching in the casethat silicon dioxide or silicon nitride hard etch masks are used. Thesample well may have a diameter of t approximately 50 nm, approximately100 nm, or approximately 150 nm.

Alternatively, a sample well may be fabricated using standardphotolithography processes and an alternate lift-off technique. FIG.11-3 illustrates an exemplary embodiment of forming a sample well of anintegrated device. A hard etch mask layer 11-314 is deposited onsubstrate 11-310. Hard etch mask layer 11-314 may include Ti or anyother suitable material. Substrate 11-310 may include a dielectric(e.g., SiO₂) or any other suitable material. A layer of ARC 11-322 isthen deposited onto the hard etch mask layer 11-314 followed byphotoresist layer 11-320. Conventional photolithographic techniques areused to pattern the photoresist such that a pillar 11-320 of resist isformed. This photoresist pillar pattern is used as an etch mask foretching the ARC layer 11-322 and hard etch mask layer 11-314.Photoresist layer 11-320 and ARC 11-322 is then stripped, and pillar11-330 of hard etch mask is left behind. Conventional techniques may beused to dissolve the remaining photoresist and the ARC layer off thepillar. The following step may involve depositing layer 11-312 directlyon top of pillar 11-330 creating a capped pillar. To form the samplewell, the capped pillar is stripped by a hydrogen peroxide etch, orother suitable etch, which erodes layer 11-314, “lifts off” the cap andresults in a sample well in layer 11-312.

In some embodiments, the sample well may be fabricated to attenuateplasmon transmission through the sample well in any suitable way. Forexample, the sample well may be fabricated in a multi-layered stack. Themulti-layered stack may include, but is not limited to, a metal layerdeposited on a substrate, an absorbing layer and/or a surface layer. Thesurface layer may be a passivation layer. The multi-layer stack may befabricated in any suitable way. Conventional patterning and etchingtechniques may be used. A metal layer may be deposited onto a substrate.Alternatively, an absorbing layer may be deposited onto the metal layer.Alternatively, a surface passivation layer may be deposited onto theabsorbing layer/metal layer stack. A photoresist and anti-reflectionlayer may be deposited onto the top layer of the multilayer stack. Thephotoresist layer may be patterned with the dimensions of the samplewell. The multi-layer stack may be directly etched to form the samplewell.

The absorbing layers may include any suitable absorbing materials.Non-limiting examples include silicon nitride, TiN, aSi, TaN, Ge and/orCr. Variants of the named materials are also possible, such as Si₃N₄.The metal layer and the surface layer may be made of any suitablematerials. For example, Al, AlSi, or AlCu may be used for the metallayer. The surface layer may be made of Al or Al₂O₃, for example. Thesample well in the multilayer stack may be fabricating using theprocesses described above.

Additionally and/or alternatively, a reflective layer may be depositeddirectly on top of the substrate before depositing the multi-layeredstack to control the focus of the light beam during photolithography. Areflective layer may be deposited directly on top of the substrate andpatterned with the dimensions of the sample well. Optionally, a layer ofanti-reflection coating followed by a layer of photoresist may bedeposited on top of the patterned reflective coating layer and patternedto leave a pillar of ARC and photoresist at a location on the substrate.A multilayer stack may then be deposited on top of the pillar,reflective layer, and substrate. The capped pillar may be removed usinga lift-off process, as described above, forming a sample well at thelocation of the substrate where the piller had been.

Similarly, a Ti pillar may be used to create the sample well. The firststep may involve depositing a layer of Ti on the substrate, followed bya layer of anti-reflection coating and a layer of photoresist. The Tilayer may be patterned and etched to form a Ti pillar. The multilayerstack may be deposited on top of the Ti pillar and substrate. Finally,the Ti pillar may be removed forming a sample well at a location of thesubstrate corresponding to where the Ti pillar had been.

Any suitable deposition methods may be used. For example, PVD, CVD,sputtering, ALD, e-beam deposition and/or thermal evaporation may beused to deposit one or more layers. The deposition environment may becontrolled to prevent oxidation of the layers in between depositions.For example, the environment may be kept at a high vacuum and/orlow-oxygen state during and between depositions of one or more layers.

B. Concentric Grating (Bullseye) Fabrication Process

A concentric grating, or bullseye, may be fabricated in any suitableway. In some embodiments, a concentric grating may be fabricated usingstandard photolithography processes and etching techniques. Any suitabledielectric material, such as SiO₂ or silicon nitride, may be used toform the concentric grating. In the embodiment illustrated in FIG. 11-4,a SiO₂ layer 11-1010 is used to make the concentric grating. The firststep in the fabrication process may involve depositing a hard etch mask11-1014 directly on top of the SiO₂ layer. The next step (act 11-1001)in the fabrication process may involve depositing a photoresist layer11-1020 directly on top of an anti-reflection coating layer 11-1022 ontothe hard etch mask. Conventional photolithographic techniques are usedto create (act 11-1003 and act 11-1005) the bullseye pattern in the hardetch mask. The bullseye pattern is then transferred (11-1007) to theunderlying SiO₂ layer using etching, for example reactive ion etchingtechniques to form the concentric grating. The thickness of theconcentric grating can be any suitable thickness. In the embodimentillustrated in FIG. 11-4, the etch depth is approximately 80 nm.Conventional techniques are used to strip (act 11-1009) the resist andetch mask residues and clean the surface of the concentric grating. Thenanoaperture in layer 11-1012 may be fabricated (act 11-1011) directlyon top of the concentric grating using the lift-off or etch processes.In other embodiments, other layers may be deposited between theconcentric grating and the nanoaperture.

Alternatively, in some embodiments, the nanoaperture may be positionedcentral to the concentric grating. This precise alignment of thenanoaperture may be achieved in any suitable way. In the embodimentillustrated in FIG. 11-5, positioning of the nanoaperture is achievedusing a self-aligned fabrication process. The first step may involveforming the concentric grating according to the techniques describedabove. However, in FIG. 11-5, a Ti hard etch mask 11-1114 is deposited(act 11-1101) on top of the SiO₂ substrate 11-1110. The bullseye patternis transferred to the Ti layer using etching, for example reactive ionetching (act 11-1103 and act 11-1105). A layer of resist 11-1120 and alayer of anti-reflection coating 11-1122 are deposited over the twocenter gaps in the Ti layer to cover the gaps and the center Ti pillar.The bullseye pattern is then transferred to the SiO₂ substrate usingconventional etching techniques to form the concentric grating (act11-1107). The Ti layer is then removed using an isotropic wet etch (act11-1109), for example, using peroxide, but leaving the center Ti pillar11-1116 in place. The layer of resist is then stripped usingconventional techniques. The metal nanoaperture layer is then deposited(act 11-1111) on top of the concentric grating and the Ti pillar.Lastly, the metal-capped Ti pillar is removed using a lift-off processleaving a nanoaperture precisely centered relative to the concentricgrating.

The precise alignment of the nanoaperture may be achieved in variousother ways. In the embodiment illustrated in FIG. 11-6, positioning ofthe nanoaperture is achieved using an alternate self-aligned fabricationprocess. The first step (11-1201) may involve depositing the Alnanoaperture layer 11-1212 directly on top of the SiO₂ concentricgrating substrate 11-1210. A hard etch mask 11-1214 may then bedeposited on top of the Al layer. In the embodiment illustrated in FIG.11-6, Ti is used but any material compatible with photolithographicprocesses may be used. The bullseye pattern is transferred (act 11-1203and 11-1205) to the Ti and Al layers using conventional etchingtechniques. A layer of resist 11-1220 and a layer of anti-reflectioncoating 11-1222 are deposited over the center gap in the Ti and Allayers to cover the position where the nanoaperture is to be formed. Thebullseye pattern is then transferred (act 11-1207) to the SiO₂ substrateusing conventional etching techniques to form the concentric grating. Anadditional metal layer is deposited (act 11-1209) on top of the Ti andfirst Al layer such that the metal fills the cavities in the SiO₂ layerand covers the Ti layer and the resist layer. In the embodimentillustrated in FIG. 11-6, Al is used as the additional metal layer butother suitable metals compatible with photolithographic processes may beused. Lastly, the metal-capped resist pillar 11-1230 is removed (act11-1211) using a lift-off process leaving a nanoaperture preciselycentered relative to the concentric grating.

C. Lens Fabrication Process: Refractive Lens

A refractive lens array may be created in any suitable way to improveefficiency of focusing of excitation into and collection of emissionlight from the nanoaperture. In some embodiments, a refractive lensarray may be a “gapless” array to minimize “dead zones” on the lensarray. In the embodiment illustrated in FIG. 11-7, a refractivemicrolens array is shown with no gaps between individual lenses. In someembodiments, fabricating a “gapless” array may involve two etchingsteps. A first etch (act 11-1801) may establish the depth of themicrolens topography. A second etch (act 11-1803) may follow the firstetch to eliminate the planar gaps between the individual microlensessuch that one lens stops at the edge where another lens begins. The sumof the first and second etches defines the focal length. In theembodiment illustrated in FIG. 11-8, a top view of a microlens array isshown after the first HF etch (1), after the second HF etch (2), afterthe microlens array is coated with a higher refractive index materialsilicon nitride (3), and after the high refractive index material ispolished and planarized (4).

Each refractive lens in the refractive lens array may be fabricated inany suitable way. An example refractive lens array is illustrated inFIG. 11-9 where a nanoaperture layer 11-2007 is fabricated on top of atransparent spacer layer 11-2001, which is on top of a dielectric lenslayer 11-2003, which is on top of a substrate 11-2005. In someembodiments, a refractive lens may be fabricated using standardphotolithography processes and etching techniques. Any suitabledielectric material, such as SiO₂ or silicon nitride, may be used toform the refractive lens. In the embodiment illustrated in FIG. 11-10,silicon nitride is used to fill in the SiO₂ substrate topography. Thefirst step 11-2101 in the fabrication process may involve depositing ahard etch mask directly on top of a SiO₂ substrate 11-2110. Any suitablemetal may be used for the hard etch mask 11-2114 that does not dissolveduring the same etching process used for the SiO₂ layer. For example, Cris used in FIG. 11-10, but other metals are possible. The next step mayinvolve applying a photoresist layer 11-2120 on top of the Cr hard etchmask. Conventional photolithographic techniques are used to create acircular pattern in the hard etch mask. The circular pattern is thentransferred to the underlying Cr layer using conventional etchingtechniques, such as reactive ion etching techniques, for example. TheSiO₂ layer is etched using any suitable selective etching techniquewhich can etch the SiO₂ but not the hard etch mask. For example, anisotropic wet etch using HF is used to create a concave surface in theSiO₂ layer. The Cr layer is then removed using conventional etchingtechniques. Optionally, a second wet etch using HF is performed toeliminate the gaps between lenses. To create the refractive lens, thecavity in the SiO₂ layer is filled with a high refractive index materiallayer 11-2118, such as silicon nitride. Finally, the top surface of thelens is planarized with conventional techniques, such as chemicalmechanical polishing, for example. A spacer layer 11-2124 may bedeposited on top of the silicon nitride layer. For example, a spacerlayer made of ORMOCER™ may be spun-coat on top of the silicon nitridelayer. Alternatively, a layer of SiO₂ may be deposited. The nanoaperturemay be fabricated directly on top of the refractive lens. In otherembodiments, other layers may be deposited between the refractive lensand the nanoaperture.

Alternatively, each refractive lens may include an anti-reflection layerto further improve optical efficiency. In some embodiments, ananti-reflection layer may coat a bottom, top, or all sides of a lens.First, a SiO₂ cavity 11-2210 is etched (act 11-2201) into a SiO₂ layer.In the embodiment illustrated in FIG. 11-11, an anti-reflection layer11-2222 is deposited (act 11-2203) on the etched SiO₂ cavity 11-2210before the cavity is filled (act 11-2205) with a silicon nitride layer11-2218. The silicon nitride layer is polished (act 11-2207) via CMP anda second antireflection layer 11-2226 is deposited (act 11-2209) on topof the polished silicon nitride layer. Additional layers may bedeposited on top of the antireflection layer, such as the spacer layerdescribed above and shown as layer 11-2224 in FIG. 11-11. Theanti-reflection layers may have the following parameters: index ofrefraction, n_(C)=sqrt(n_(oxide), n_(nitride))=sqrt(1.46*1.91)=1.67;range of refractive index from 1.67 to 1.75; and, thicknesst=λ/(4*n_(C))=675 nm/(1.670*4)=101.1 nm. The anti-reflection layer maybe deposited in any suitable way. For example, PECVD may be used.Alternatively, LPCVD may be used.

D. Lens Fabrication Process: Fresnel Lens

A diffractive optical element (DOE) may have any suitable shape and maybe fabricated in any suitable way to improve the focusing ofluminescence on the CMOS sensors and the sorting of the luminescencephotons. In some embodiments, the DOE may include a section of a Fresnellens. As illustrated in FIG. 11-12 to 11-19, the DOE 11-2301 ischaracterized as a square section offset from the center of a Fresnellens. As illustrated in FIG. 11-13, the DOE may comprise two unit celllayers where the first layer 11-2401 contains “small” features and thesecond layer 11-2403 contains “large” features. The unit cell layers mayhave any suitable pitch, and may further have varying pitch according tothe optical design of the Fresnel lens. As illustrated in the example inFIG. 11-13, the small DOE layer has a pitch of 220 nm and the large DOElayer has a pitch of 440 nm. The large DOE layer may be overlaid ontothe small DOE layer (or vice versa) to create a multilevel diffractiveoptic. FIG. 11-13 illustrates an example of an offset Fresnel array11-2405 where large fiducial markers surround the offset Fresnel lens.Additionally, the offset Fresnel array may be positioned on top of thesensor to provide focusing and spectral separation of luminescence intothe sensor.

Alternatively, a diffractive optical element (DOE) may be embeddedunderneath the nanoaperture to improve the focusing of the excitationenergy into and collection of luminescence from the nanoaperture. Insome embodiments, the embedded Fresnel lens positioned underneath thenanoaperture and the offset Fresnel lens positioned over the sensor mayhave a tiered structure with a variable period and variable step size.In other embodiments, only the offset Fresnel lens positioned over thesensor may have a variable period and variable step size. Thesediffractive lenses may be fabricated using standard photolithographyprocesses and etching techniques. As illustrated in FIG. 11-14, thediffractive lens pattern 11-2501 is characterized as having a tieredstructure comprising large steps (large pattern) and small steps (smallpattern) on each larger step, both having a decreasing period whenviewed from left to right. The fabrication process for thevariable-period stepped diffractive lens may involve etching the largesteps first, followed by etching the small steps as illustrated in FIG.11-16, which may protect the corners of the large steps during thesecond etch. An alternate approach is to etch the small steps first on aflat substrate, followed by etching the large steps, as illustrated inFIG. 11-15. Any suitable dielectric material, such as SiO₂ or siliconnitride, TiO₂ or Ta₂O₅, may be used to form the fill-in layer for thediffractive lens and the tiered layer. In the embodiment illustrated inFIG. 11-15, silicon nitride is used to make the fill-in layer and SiO₂is used to make the tiered layer.

The first step in the fabrication process of the tiered SiO₂ layer mayinvolve depositing a hard etch mask 11-2614 directly on top of a SiO₂layer 11-2610 followed by an anti-reflection layer 11-2622 followed by aphotoresist layer 11-2620. Any suitable material may be used for thehard etch mask. For example, a-Si may be used for the hard etch maskshown in FIG. 11-15, but other materials are possible. The next step mayinvolve applying an ARC and/or a photoresist layer on top of the a-Sihard etch mask. Conventional photolithographic techniques may be used tocreate the variable-period large binary pattern. The patterns aretransferred to the underlying Si layer using conventional etchingtechniques, such as reactive ion etching techniques, for example.

The etch depth of a large diffractive lens step can be any suitabledepth that accomplishes the desired focal length. In the embodimentillustrated in FIG. 11-16, this etch depth into the SiO₂ layer isapproximately 684 nm for the large step. Conventional techniques arethen used to strip (act 11-2605) the resist and etch mask residues andclean the surface of the SiO₂ layer. The next step may involve etchingthe small steps on each large step. In the embodiment illustrated inFIG. 11-16, each large steps comprises four smaller steps.

A second Si hard etch mask 11-2644 is then deposited on the patternedSiO₂ layer 11-2610. An ARC layer 11-2642 is then deposited on top of theSi layer 11-2610 followed by a photoresist etch mask layer 11-2640. Thesecond variable-period small binary pattern is transferred to thephotoresist and/or the ARC layer. In the embodiment illustrated in FIG.11-16, the fabrication steps are similar to that described in FIG.11-15, however, two small steps are etched per large step leaving foursteps in total. In other embodiments, any number of steps may be used.The small steps are then etched into the SiO₂ layer 11-2710. Thethickness of a small diffractive lens step can be any suitablethickness. In the embodiment illustrated in FIG. 11-16, the etch depthinto the SiO₂ layer is approximately 342 nm for the small step.Conventional techniques are then used to strip the resist and clean thesurface of the SiO₂ layer.

Additional stages in the fabrication process following creation of thetiered SiO₂ layer 11-2810 may involve filling the cavities with anysuitable high index lens material 11-2818, such as silicon nitride, forexample, to create an “embedded Fresnel lens”, as illustrated in FIG.11-17 to 11-18. The tiered structure used for the “embedded Fresnellens” may have approximately the same and/or smaller size features asthe tiered structure used for the offset Fresnel lens. Any method ofdepositing the silicon nitride may be used such as PECVD, for example.Optionally, the silicon nitride layer may be uniformly polished downuntil the top step of the SiO₂ material is exposed. Alternatively, thesilicon nitride layer 11-2818 is uniformly polished but the SiO₂material is not exposed. In the embodiment illustrated in FIG. 11-18, asecond layer 11-2928 of SiO₂ is then deposited via PECVD on top of thepolished silicon nitride layer 11-2918 and polished via CMP. In someembodiments, the spacer layer 11-2928 may have a thickness equal to thefocal length in that spacer layer material. Additionally, other suitabletransparent spacer layers may be deposited on top of the silicon nitridelayer. The nanoaperture layer may then be fabricated on top of thetransparent spacer layer and/or additional layers.

Alternatively, in the embodiment illustrated in FIG. 11-19, the tieredlayer 11-3018 for the diffractive lens is made of silicon nitride. Thesilicon nitride layer 11-3018 may be deposited at any suitable thicknesson top of substrate 11-3010 followed by etch mask 11-3014, ARC layer11-3022 and photoresist layer 11-3020. In the embodiment illustrated inFIG. 11-19, the silicon nitride layer is approximately 1 um thick. Thefabrication processes may be similar to the one described above inregards to creating the tiered, variable-period diffractive lens layerin SiO₂. Optionally, a different hard mask may be used to create thesilicon nitride tiered layer. The silicon nitride tiered layer may haveapproximately the same and/or smaller size features as the SiO₂ tieredlayer. After the silicon nitride tiered layer is made, the siliconnitride layer may be coated in any suitable dielectric material 11-3028.In the embodiment illustrated in FIG. 11-19, the silicon nitride layeris coated with SiO₂ layer 11-3028. The SiO₂ layer may be deposited usingconventional deposition processes such as PECVD, for example. The SiO₂layer may then be polished to create a flat, planar surface. Thenanoaperture layer may then be fabricated on top of the SiO₂ layerand/or additional layers.

Certain features of the diffractive optic may require a certain degreeof uniformity and/or accuracy during the fabrication process to yield astructure with the desired optical properties. For example, the etchdepth of the large and small steps may require a certain degree ofaccuracy. In some embodiments, an etch depth within 50 or 10% of thetarget may be required to achieve the desired power efficiency into thefocal spot nanoaperture. Additionally, the etching of the lens featuresmay require a certain degree of uniformity. For example, etch uniformitywithin 5% (or 50 nm) is required to achieve the desired focal length.

Any of the lenses described above may be fabricated using any suitabledeposition and etching processes to create improved optical properties.By way of example and not limitation, PECVD may be used. The depositionparameters may be tuned in any suitable way to reduce autoluminescence,reduce lens absorption of luminescence, and/or create a high index ofrefraction. For example, a reduced autoluminescence and lens absorptionmay be achieved by reducing the density of Si—Si bonds, which may formsilicon nanocrystals, during deposition of silicon nitride. In someembodiments, the input gases and their ratios may be modified to reducethe density of Si—Si bonds and silicon nanocrystals. For example, SiH₄and N₂ may be used and their ratios adjusted in any suitable way toreduce the density of Si nanocrystals. In other embodiments, SiH₄ andNH₃ may be used and their ratios adjusted in any suitable way to reducethe density of Si—Si bonds and silicon nanocrystals. For example, theratio of NH₃ to SiH₄ may be at least 10:1. Additionally, tuning thefrequencies that control the plasma during PECVD may be used to improveoptical properties. For example, the low frequency (e.g. below 0.5 MHz)to high frequency (e.g. above 10 MHz) ratio may be at least 1:1.

Additionally, the above described deposition parameters may tune thelens index of refraction to improve optical properties. In someembodiments, the index of refraction for a silicon nitride lens may beless than n=1.92 and associated with a wavelength of 633 nm for asuitable low autoluminescence effect and/or a suitable low absorptionloss. The tuned qualities described above may be related to,proportional, correlated, associated with and/or dependent each other.For example, an index of refraction of n=1.92 is indicative of a lowluminescence and low absorption loss which is related to a low densityof Si—Si bonds and silicon nanocrystals for a lens made of siliconnitride.

X. CONCLUSION

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated that various alterations,modifications, and improvements will readily occur to those skilled inthe art.

Such alterations, modifications, and improvements are intended to bepart of this disclosure, and are intended to be within the spirit andscope of the invention. Further, though advantages of the presentinvention are indicated, it should be appreciated that not everyembodiment of the invention will include every described advantage. Someembodiments may not implement any features described as advantageousherein and in some instances. Accordingly, the foregoing description anddrawings are by way of example only.

The above-described embodiments of the present invention can beimplemented in any of numerous ways. For example, the embodiments may beimplemented using hardware, software or a combination thereof. Whenimplemented in software, the software code can be executed on anysuitable processor or collection of processors, whether provided in asingle computer or distributed among multiple computers. Such processorsmay be implemented as integrated circuits, with one or more processorsin an integrated circuit component, including commercially availableintegrated circuit components known in the art by names such as CPUchips, GPU chips, microprocessor, microcontroller, or co-processor.Alternatively, a processor may be implemented in custom circuitry, suchas an ASIC, or semicustom circuitry resulting from configuring aprogrammable logic device. As yet a further alternative, a processor maybe a portion of a larger circuit or semiconductor device, whethercommercially available, semicustom or custom. As a specific example,some commercially available microprocessors have multiple cores suchthat one or a subset of those cores may constitute a processor. Though,a processor may be implemented using circuitry in any suitable format.

Further, it should be appreciated that a computer may be embodied in anyof a number of forms, such as a rack-mounted computer, a desktopcomputer, a laptop computer, or a tablet computer. Additionally, acomputer may be embedded in a device not generally regarded as acomputer but with suitable processing capabilities, including a PersonalDigital Assistant (PDA), a smart phone or any other suitable portable orfixed electronic device.

Also, a computer may have one or more input and output devices. Thesedevices can be used, among other things, to present a user interface.Examples of output devices that can be used to provide a user interfaceinclude printers or display screens for visual presentation of outputand speakers or other sound generating devices for audible presentationof output. Examples of input devices that can be used for a userinterface include keyboards, and pointing devices, such as mice, touchpads, and digitizing tablets. As another example, a computer may receiveinput information through speech recognition or in other audible format.

Such computers may be interconnected by one or more networks in anysuitable form, including as a local area network or a wide area network,such as an enterprise network or the Internet. Such networks may bebased on any suitable technology and may operate according to anysuitable protocol and may include wireless networks, wired networks orfiber optic networks.

Also, the various methods or processes outlined herein may be coded assoftware that is executable on one or more processors that employ anyone of a variety of operating systems or platforms. Additionally, suchsoftware may be written using any of a number of suitable programminglanguages and/or programming or scripting tools, and also may becompiled as executable machine language code or intermediate code thatis executed on a framework or virtual machine.

In this respect, the invention may be embodied as a computer readablestorage medium (or multiple computer readable media) (e.g., a computermemory, one or more floppy discs, compact discs (CD), optical discs,digital video disks (DVD), magnetic tapes, flash memories, circuitconfigurations in Field Programmable Gate Arrays or other semiconductordevices, or other tangible computer storage medium) encoded with one ormore programs that, when executed on one or more computers or otherprocessors, perform methods that implement the various embodiments ofthe invention discussed above. As is apparent from the foregoingexamples, a computer readable storage medium may retain information fora sufficient time to provide computer-executable instructions in anon-transitory form. Such a computer readable storage medium or mediacan be transportable, such that the program or programs stored thereoncan be loaded onto one or more different computers or other processorsto implement various aspects of the present invention as discussedabove. As used herein, the term “computer-readable storage medium”encompasses only a computer-readable medium that can be considered to bea manufacture (i.e., article of manufacture) or a machine. Alternativelyor additionally, the invention may be embodied as a computer readablemedium other than a computer-readable storage medium, such as apropagating signal.

The terms “program” or “software” are used herein in a generic sense torefer to any type of computer code or set of computer-executableinstructions that can be employed to program a computer or otherprocessor to implement various aspects of the present invention asdiscussed above. Additionally, it should be appreciated that accordingto one aspect of this embodiment, one or more computer programs thatwhen executed perform methods of the present invention need not resideon a single computer or processor, but may be distributed in a modularfashion amongst a number of different computers or processors toimplement various aspects of the present invention.

Computer-executable instructions may be in many forms, such as programmodules, executed by one or more computers or other devices. Generally,program modules include routines, programs, objects, components, datastructures, etc. that perform particular tasks or implement particularabstract data types. Typically the functionality of the program modulesmay be combined or distributed as desired in various embodiments.

Also, data structures may be stored in computer-readable media in anysuitable form. For simplicity of illustration, data structures may beshown to have fields that are related through location in the datastructure. Such relationships may likewise be achieved by assigningstorage for the fields with locations in a computer-readable medium thatconveys relationship between the fields. However, any suitable mechanismmay be used to establish a relationship between information in fields ofa data structure, including through the use of pointers, tags or othermechanisms that establish relationship between data elements.

Various aspects of the present invention may be used alone, incombination, or in a variety of arrangements not specifically discussedin the embodiments described in the foregoing and is therefore notlimited in its application to the details and arrangement of componentsset forth in the foregoing description or illustrated in the drawings.For example, aspects described in one embodiment may be combined in anymanner with aspects described in other embodiments.

Also, the invention may be embodied as a method, of which an example hasbeen provided. The acts performed as part of the method may be orderedin any suitable way. Accordingly, embodiments may be constructed inwhich acts are performed in an order different than illustrated, whichmay include performing some acts simultaneously, even though shown assequential acts in illustrative embodiments.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

Also, the phraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of“including,” “comprising,” or “having,” “containing,” “involving,” andvariations thereof herein, is meant to encompass the items listedthereafter and equivalents thereof as well as additional items.

What is claimed is:
 1. An instrument configured to interface with anassay chip comprising a plurality of sample wells, each sample well ofthe plurality of sample wells configured to receive a sample, theinstrument comprising: at least one pulsed excitation light sourceconfigured to excite the sample of at least a portion of the pluralityof sample wells; a plurality of sensors, each sensor of the plurality ofsensors corresponding to a sample well of the plurality of sample wells,wherein each sensor of the plurality of sensors is configured to detectemission energy from the sample in a respective sample well, whereineach sensor of the plurality of sensors is capable of detecting thedetection time of the emission energy; and at least one optical elementconfigured to direct the emission energy from each sample well of theplurality of sample wells towards a respective sensor of the pluralityof sensors.